Microorganisms and methods for enhancing the availability of reducing equivalents in the presence of methanol, and for producing 1,2-propanediol, n-propanol, 1,3-propanediol, or glycerol related thereto

ABSTRACT

Provided herein is a non-naturally occurring microbial organism having a methanol metabolic pathway that can enhance the availability of reducing equivalents in the presence of methanol. Such reducing equivalents can be used to increase the product yield of organic compounds produced by the microbial organism, such as 1,2-propanediol, n-propanol, 1,3-propanediol or glycerol. Also provided herein are methods for using such an organism to produce 1,2-propanediol, n-propanol, 1,3-propanediol or glycerol.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Ser. No. 61/766,635 filedFeb. 19, 2013, and U.S. Ser. No. 61/722,629 filed Nov. 5, 2012, each ofwhich is incorporated herein by reference in its entirety.

1. SUMMARY

Provided herein are methods generally relating to metabolic andbiosynthetic processes and microbial organisms capable of producingorganic compounds. Specifically, provided herein is a non-naturallyoccurring microbial organism having a methanol metabolic pathway thatcan enhance the availability of reducing equivalents in the presence ofmethanol. Such reducing equivalents can be used to increase the productyield of organic compounds produced by the microbial organism, such as1,2-propanediol, n-propanol, 1,3-propanediol and/or glycerol. Alsoprovided herein are non-naturally occurring microbial organisms andmethods thereof to produce optimal yields of 1,2-propanediol,n-propanol, 1,3-propanediol and/or glycerol.

In a first aspect, provided herein is a non-naturally occurringmicrobial organism having a methanol metabolic pathway, wherein saidorganism comprises at least one exogenous nucleic acid encoding amethanol metabolic pathway enzyme expressed in a sufficient amount toenhance the availability of reducing equivalents in the presence ofmethanol. In certain embodiments, the methanol metabolic pathwaycomprises one or more enzymes selected from the group consisting of amethanol methyltransferase; a methylenetetrahydrofolate reductase; amethylenetetrahydrofolate dehydrogenase; a methenyltetrahydrofolatecyclohydrolase; a formyltetrahydrofolate deformylase; aformyltetrahydrofolate synthetase; a formate hydrogen lyase; ahydrogenase; a formate dehydrogenase; a methanol dehydrogenase; aformaldehyde activating enzyme; a formaldehyde dehydrogenase; aS-(hydroxymethyl)glutathione synthase; a glutathione-dependentformaldehyde dehydrogenase; and an S-formylglutathione hydrolase. Suchorganisms advantageously allow for the production of reducingequivalents, which can then be used by the organism for the productionof 1,2-propanediol, n-propanol, 1,3-propanediol or glycerol using anyone of the 1,2-propanediol, n-propanol, 1,3-propanediol or glycerolpathways, respectively, provided herein.

In a second aspect, provided herein is a non-naturally occurringmicrobial organism having (1) a methanol metabolic pathway, wherein saidorganism comprises at least one exogenous nucleic acid encoding amethanol metabolic pathway enzyme expressed in a sufficient amount toenhance the availability of reducing equivalents in the presence ofmethanol; and (2) a 1,2-propanediol pathway, wherein said organismcomprises at least one exogenous nucleic acid encoding a 1,2-propanediolpathway enzyme expressed in a sufficient amount to produce1,2-propanediol. In certain embodiments, the 1,2-propanediol pathwayenzyme is selected from the group consisting of a methylglyoxalsynthase; a methylglyoxal reductase (acetol-forming); an acetolreductase; a methylglyoxal reductase (lactaldehyde-forming); and alactaldehyde reductase.

In a third aspect, provided herein is a non-naturally occurringmicrobial organism having (1) a methanol metabolic pathway, wherein saidorganism comprises at least one exogenous nucleic acid encoding amethanol metabolic pathway enzyme expressed in a sufficient amount toenhance the availability of reducing equivalents in the presence ofmethanol; and (2) a n-propanol pathway, wherein said organism comprisesat least one exogenous nucleic acid encoding a n-propanol pathway enzymeexpressed in a sufficient amount to produce n-propanol. In certainembodiments, the n-propanol pathway enzyme is selected from the groupconsisting of a methylglyoxal synthase; a methylglyoxal reductase(acetol-forming); an acetol reductase; a methylglyoxal reductase(lactaldehyde-forming); a lactaldehyde reductase; a 1,2-propanedioldehydratase; and a propanal reductase.

In a fourth aspect, provided herein is a non-naturally occurringmicrobial organism having (1) a methanol metabolic pathway, wherein saidorganism comprises at least one exogenous nucleic acid encoding amethanol metabolic pathway enzyme expressed in a sufficient amount toenhance the availability of reducing equivalents in the presence ofmethanol; and (2) a 1,3-propanediol pathway, wherein said organismcomprises at least one exogenous nucleic acid encoding a 1,3-propanediolpathway enzyme expressed in a sufficient amount to produce1,3-propanediol. In certain embodiments, the 1,3-propanediol pathwayenzyme is selected from the group consisting of aglyceraldehyde-3-phosphate reductase; a glycerol-3-phosphate phosphataseor a glycerol kinase; a glycerol dehydratase; a 3-hydroxypropanalreductase; a dihydroxyacetone phosphate phosphatase or adihydroxyacetone kinase; a dihydroxyacetone reductase; and adihydroxyacetone phosphate reductase.

In a fifth aspect, provided herein is a non-naturally occurringmicrobial organism having (1) a methanol metabolic pathway, wherein saidorganism comprises at least one exogenous nucleic acid encoding amethanol metabolic pathway enzyme expressed in a sufficient amount toenhance the availability of reducing equivalents in the presence ofmethanol; and (2) a glycerol pathway, wherein said organism comprises atleast one exogenous nucleic acid encoding a glycerol pathway enzymeexpressed in a sufficient amount to produce glycerol. In certainembodiments, the glycerol pathway enzyme is selected from the groupconsisting of a glyceraldehyde-3-phosphate reductase; aglycerol-3-phosphate phosphatase or a glycerol kinase; adihydroxyacetone phosphate phosphatase or a dihydroxyacetone kinase; adihydroxyacetone reductase; and a dihydroxyacetone phosphate reductase.

In other embodiments, the organism having a methanol metabolic pathway,either alone or in combination with a 1,2-propanediol, n-propanol,1,3-propanediol or glycerol pathway, as provided herein, furthercomprises a formaldehyde assimilation pathway that utilizesformaldehyde, e.g., obtained from the oxidation of methanol, in theformation of intermediates of certain central metabolic pathways thatcan be used, for example, in the formation of biomass. In some ofembodiments, the formaldehyde assimilation pathway comprises ahexylose-6-phosphate synthase, 6-phospho-3-hexyloisomerase,dihydroxyacetone synthase or dihydroxyacetone kinase. In certainembodiments, provided herein is a non-naturally occurring microbialorganism having a methanol metabolic pathway, wherein said organismcomprises at least one exogenous nucleic acid encoding a methanoldehydrogenase expressed in a sufficient amount to enhance theavailability of reducing equivalents in the presence of methanol and/orexpressed in a sufficient amount to convert methanol to formaldehyde. Insome embodiments, the microbial organism further comprises aformaldehyde assimilation pathway. In certain embodiments, the organismfurther comprises at least one exogenous nucleic acid encoding aformaldehyde assimilation pathway enzyme expressed in a sufficientamount to produce an intermediate of glycolysis. In certain embodiments,the formaldehyde assimilation pathway enzyme is selected from the groupconsisting of a hexylose-6-phosphate synthase,6-phospho-3-hexyloisomerase, dihydroxyacetone synthase anddihydroxyacetone kinase.

In some embodiments, the organism further comprises one or more genedisruptions, occurring in one or more endogenous genes encodingprotein(s) or enzyme(s) involved in native production of ethanol,glycerol, acetate, lactate, formate, CO₂, and/or amino acids by saidmicrobial organism, wherein said one or more gene disruptions conferincreased production of 1,2-propanediol, n-propanol, 1,3-propanediol orglycerol in said microbial organism. In some embodiments, one or moreendogenous enzymes involved in native production of ethanol, glycerol,acetate, lactate, formate, CO₂ and/or amino acids by the microbialorganism, has attenuated enzyme activity or expression levels. Incertain embodiments, the organism comprises from one to twenty-five genedisruptions. In other embodiments, the organism comprises from one totwenty gene disruptions. In some embodiments, the organism comprisesfrom one to fifteen gene disruptions. In other embodiments, the organismcomprises from one to ten gene disruptions. In some embodiments, theorganism comprises from one to five gene disruptions. In certainembodiments, the organism comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 genedisruptions or more.

In other aspects, provided herein are methods for producing1,2-propanediol, n-propanol, 1,3-propanediol or glycerol, comprisingculturing any one of the non-naturally occurring microbial organismscomprising a methanol metabolic pathway and a 1,2-propanediol,n-propanol, 1,3-propanediol or glycerol pathway provided herein underconditions and for a sufficient period of time to produce1,2-propanediol, n-propanol, 1,3-propanediol or glycerol. In certainembodiments, the organism is cultured in a substantially anaerobicculture medium. In one embodiment, provided herein is a method forproducing 1,2-propanediol, comprising culturing any one of thenon-naturally occurring microbial organisms comprising a methanolmetabolic pathway and a 1,2-propanediol pathway provided herein underconditions and for a sufficient period of time to produce1,2-propanediol. In another embodiment, provided herein is a method forproducing n-propanol, comprising culturing any one of the non-naturallyoccurring microbial organisms comprising a n-propanol pathway providedherein under conditions and for a sufficient period of time to producen-propanol. In another embodiment, provided herein is a method forproducing 1,3-propanediol, comprising culturing any one of thenon-naturally occurring microbial organisms comprising a methanolmetabolic pathway and a 1,3-propanediol pathway provided herein underconditions and for a sufficient period of time to produce1,3-propanediol. In yet another embodiment, provided herein is a methodfor producing glycerol, comprising culturing any one of thenon-naturally occurring microbial organisms comprising a methanolmetabolic pathway and a glycerol pathway provided herein underconditions and for a sufficient period of time to produce glycerol.

2. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows exemplary metabolic pathways enabling the extraction ofreducing equivalents from methanol. The enzymatic transformations shownare carried out by the following enzymes: 1A) a methanolmethyltransferase, 1B) a methylenetetrahydrofolate reductase, 1C) amethylenetetrahydrofolate dehydrogenase, 1D) a methenyltetrahydrofolatecyclohydrolase, 1E) a formyltetrahydrofolate deformylase, 1F) aformyltetrahydrofolate synthetase, 1G) a formate hydrogen lyase, 1H) ahydrogenase, 1I) a formate dehydrogenase, 1J) a methanol dehydrogenase,1K) a formaldehyde activating enzyme, 1L) a formaldehyde dehydrogenase,1M) a S-(hydroxymethyl)glutathione synthase, 1N) a glutathione-dependentformaldehyde dehydrogenase, and 1O) a S-formylglutathione hydrolase. Incertain embodiments, steps K and/or M are spontaneous.

FIG. 2 shows exemplary 1,2-propanediol and n-propanol pathways, whichcan be used to increase 1,2-propanediol or n-propanol, respectively,from carbohydrates when reducing equivalents produced by a methanolmetabolic pathway provided herein are available. The enzymatictransformations shown are carried out by the following enzymes: 2A) amethylglyoxal synthase; 2B) a methylglyoxal reductase (acetol-forming);2C) an acetol reductase; 2D) a methylglyoxal reductase(lactaldehyde-forming); 2E) a lactaldehyde reductase; 2F) a1,2-propanediol dehydratase; and 2G) a propanal reductase.1,2-propanediol production can be carried out by 2A, 2B and 2C; or 2A,2D and 2E. n-Propanol production can be carried out by 2A, 2B, 2C, 2Fand 2G; or 2A, 2D, 2E, 2F and 2G.

FIG. 3 shows exemplary 1,3-propanediol and glycerol pathways, which canbe used to increase 1,3-propanediol or glycerol, respectively, fromcarbohydrates when reducing equivalents produced by a methanol metabolicpathway provided herein are available. The enzymatic transformationsshown are carried out by the following enzymes: 3A) aglyceraldehyde-3-phosphate reductase; 3B) a glycerol-3-phosphatephosphatase or a glycerol kinase; 3C) a glycerol dehydratase; 3D) a3-hydroxypropanal reductase; 3E) a dihydroxyacetone phosphatephosphatase or a dihydroxyacetone kinase: 3F) a dihydroxyacetonereductase; and 3G) a dihydroxyacetone phosphate reductase.1,3-propanediol production can be carried out by 3A, 3B, 3C and 3D; 3G,3B, 3C and 3D; or 3E, 3F, 3C and 3D. Glycerol production can be carriedout by 3A and 3B; 3G and 3B; or 3E and 3F.

FIG. 4 shows an exemplary formaldehyde assimilation pathway. Theenzymatic transformations are carried out by the following enzymes: 3A)a hexylose-6-phosphate synthase, and 3B) a 6-phospho-3-hexyloisomerase.

FIG. 5 shows an exemplary formaldehyde assimilation pathway. Theenzymatic transformations are carried out by the following enzymes: 4A)a dihydroxyacetone synthase, and 4B) a dihydroxyacetone kinase.

3. DETAILED DESCRIPTION OF THE INVENTION 3.1 Definitions

As used herein, the term “non-naturally occurring” when used inreference to a microbial organism or microorganism of the invention isintended to mean that the microbial organism has at least one geneticalteration not normally found in a naturally occurring strain of thereferenced species, including wild-type strains of the referencedspecies. Genetic alterations include, for example, modificationsintroducing expressible nucleic acids encoding metabolic polypeptides,other nucleic acid additions, nucleic acid deletions and/or otherfunctional disruption of the microbial organism's genetic material. Suchmodifications include, for example, coding regions and functionalfragments thereof, for heterologous, homologous or both heterologous andhomologous polypeptides for the referenced species. Additionalmodifications include, for example, non-coding regulatory regions inwhich the modifications alter expression of a gene or operon. Exemplarymetabolic polypeptides include enzymes or proteins within a1,2-propanediol, n-propanol, 1,3-propanediol or glycerol biosyntheticpathway.

As used herein, “1,2-propanediol” (also known as propylene glycol; IUPACname propane 1,2-diol) has the chemical formula HO—CH₂—CHOH—CH₃. Thechemical structure of 1,2-propanediol is shown below:

As used herein, “n-propanol” (also known as propan-1-ol, 1-propylalcohol, n-propyl alcohol, 1-propanol, or simply propanol; IUPAC namepropan-1-ol) has the chemical formula CH₃CH₂CH₂OH. The chemicalstructure of n-propanol is shown below:

As used herein, “1,3-propanediol” (IUPAC name propane-1,3-diol) has theformula CH₂(CH₂OH)₂. The chemical structure of 1,3-propanediol is shownbelow:

As used herein, “glycerol” (also known as glycerine or glycerin; IUPACname propane-1,2,3-triol) is a simple polyol compound. The chemicalstructure of glycerol is shown below:

A metabolic modification refers to a biochemical reaction that isaltered from its naturally occurring state. Therefore, non-naturallyoccurring microorganisms can have genetic modifications to nucleic acidsencoding metabolic polypeptides, or functional fragments thereof.Exemplary metabolic modifications are disclosed herein.

As used herein, the term “isolated” when used in reference to amicrobial organism is intended to mean an organism that is substantiallyfree of at least one component as the referenced microbial organism isfound in nature. The term includes a microbial organism that is removedfrom some or all components as it is found in its natural environment.The term also includes a microbial organism that is removed from some orall components as the microbial organism is found in non-naturallyoccurring environments. Therefore, an isolated microbial organism ispartly or completely separated from other substances as it is found innature or as it is grown, stored or subsisted in non-naturally occurringenvironments. Specific examples of isolated microbial organisms includepartially pure microbes, substantially pure microbes and microbescultured in a medium that is non-naturally occurring.

As used herein, the terms “microbial,” “microbial organism” or“microorganism” are intended to mean any organism that exists as amicroscopic cell that is included within the domains of archaea,bacteria or eukarya. Therefore, the term is intended to encompassprokaryotic or eukaryotic cells or organisms having a microscopic sizeand includes bacteria, archaea and eubacteria of all species as well aseukaryotic microorganisms such as yeast and fungi. The term alsoincludes cell cultures of any species that can be cultured for theproduction of a biochemical.

As used herein, the term “CoA” or “coenzyme A” is intended to mean anorganic cofactor or prosthetic group (nonprotein portion of an enzyme)whose presence is required for the activity of many enzymes (theapoenzyme) to form an active enzyme system. Coenzyme A functions incertain condensing enzymes, acts in acetyl or other acyl group transferand in fatty acid synthesis and oxidation, pyruvate oxidation and inother acetylation.

As used herein, the term “substantially anaerobic” when used inreference to a culture or growth condition is intended to mean that theamount of oxygen is less than about 10% of saturation for dissolvedoxygen in liquid media. The term also is intended to include sealedchambers of liquid or solid medium maintained with an atmosphere of lessthan about 1% oxygen.

As used herein, the term “gene disruption,” or grammatical equivalentsthereof, is intended to mean a genetic alteration that renders theencoded gene product inactive or attenuated. The genetic alteration canbe, for example, deletion of the entire gene, deletion of a regulatorysequence required for transcription or translation, deletion of aportion of the gene which results in a truncated gene product, or by anyof various mutation strategies that inactivate or attenuate the encodedgene product. One particularly useful method of gene disruption iscomplete gene deletion because it reduces or eliminates the occurrenceof genetic reversions in the non-naturally occurring microorganisms ofthe invention. The phenotypic effect of a gene disruption can be a nullmutation, which can arise from many types of mutations includinginactivating point mutations, entire gene deletions, and deletions ofchromosomal segments or entire chromosomes. Specific antisense nucleicacid compounds and enzyme inhibitors, such as antibiotics, can alsoproduce null mutant phenotype, therefore being equivalent to genedisruption.

As used herein, the term “growth-coupled” when used in reference to theproduction of a biochemical product is intended to mean that thebiosynthesis of the referenced biochemical product is produced duringthe growth phase of a microorganism. In a particular embodiment, thegrowth-coupled production can be obligatory, meaning that thebiosynthesis of the referenced biochemical is an obligatory productproduced during the growth phase of a microorganism. The term“growth-coupled” when used in reference to the consumption of abiochemical is intended to mean that the referenced biochemical isconsumed during the growth phase of a microorganism.

As used herein, the term “attenuate,” or grammatical equivalentsthereof, is intended to mean to weaken, reduce or diminish the activityor amount of an enzyme or protein. Attenuation of the activity or amountof an enzyme or protein can mimic complete disruption if the attenuationcauses the activity or amount to fall below a critical level requiredfor a given pathway to function. However, the attenuation of theactivity or amount of an enzyme or protein that mimics completedisruption for one pathway, can still be sufficient for a separatepathway to continue to function. For example, attenuation of anendogenous enzyme or protein can be sufficient to mimic the completedisruption of the same enzyme or protein for production of a fattyalcohol, fatty aldehyde or fatty acid product of the invention, but theremaining activity or amount of enzyme or protein can still besufficient to maintain other pathways, such as a pathway that iscritical for the host microbial organism to survive, reproduce or grow.Attenuation of an enzyme or protein can also be weakening, reducing ordiminishing the activity or amount of the enzyme or protein in an amountthat is sufficient to increase yield of a fatty alcohol, fatty aldehydeor fatty acid product of the invention, but does not necessarily mimiccomplete disruption of the enzyme or protein.

“Exogenous” as it is used herein is intended to mean that the referencedmolecule or the referenced activity is introduced into the hostmicrobial organism. The molecule can be introduced, for example, byintroduction of an encoding nucleic acid into the host genetic materialsuch as by integration into a host chromosome or as non-chromosomalgenetic material such as a plasmid. Therefore, the term as it is used inreference to expression of an encoding nucleic acid refers tointroduction of the encoding nucleic acid in an expressible form intothe microbial organism. When used in reference to a biosyntheticactivity, the term refers to an activity that is introduced into thehost reference organism. The source can be, for example, a homologous orheterologous encoding nucleic acid that expresses the referencedactivity following introduction into the host microbial organism.Therefore, the term “endogenous” refers to a referenced molecule oractivity that is present in the host. Similarly, the term when used inreference to expression of an encoding nucleic acid refers to expressionof an encoding nucleic acid contained within the microbial organism. Theterm “heterologous” refers to a molecule or activity derived from asource other than the referenced species whereas “homologous” refers toa molecule or activity derived from the host microbial organism.Accordingly, exogenous expression of an encoding nucleic acid of theinvention can utilize either or both a heterologous or homologousencoding nucleic acid.

It is understood that when more than one exogenous nucleic acid isincluded in a microbial organism that the more than one exogenousnucleic acids refers to the referenced encoding nucleic acid orbiosynthetic activity, as discussed above. It is further understood, asdisclosed herein, that such more than one exogenous nucleic acids can beintroduced into the host microbial organism on separate nucleic acidmolecules, on polycistronic nucleic acid molecules, or a combinationthereof, and still be considered as more than one exogenous nucleicacid. For example, as disclosed herein a microbial organism can beengineered to express two or more exogenous nucleic acids encoding adesired pathway enzyme or protein. In the case where two exogenousnucleic acids encoding a desired activity are introduced into a hostmicrobial organism, it is understood that the two exogenous nucleicacids can be introduced as a single nucleic acid, for example, on asingle plasmid, on separate plasmids, can be integrated into the hostchromosome at a single site or multiple sites, and still be consideredas two exogenous nucleic acids. Similarly, it is understood that morethan two exogenous nucleic acids can be introduced into a host organismin any desired combination, for example, on a single plasmid, onseparate plasmids, can be integrated into the host chromosome at asingle site or multiple sites, and still be considered as two or moreexogenous nucleic acids, for example three exogenous nucleic acids.Thus, the number of referenced exogenous nucleic acids or biosyntheticactivities refers to the number of encoding nucleic acids or the numberof biosynthetic activities, not the number of separate nucleic acidsintroduced into the host organism.

The non-naturally occurring microbial organisms of the invention cancontain stable genetic alterations, which refers to microorganisms thatcan be cultured for greater than five generations without loss of thealteration. Generally, stable genetic alterations include modificationsthat persist greater than 10 generations, particularly stablemodifications will persist more than about 25 generations, and moreparticularly, stable genetic modifications will be greater than 50generations, including indefinitely.

Those skilled in the art will understand that the genetic alterations,including metabolic modifications exemplified herein, are described withreference to a suitable host organism such as E. coli and theircorresponding metabolic reactions or a suitable source organism fordesired genetic material such as genes for a desired metabolic pathway.However, given the complete genome sequencing of a wide variety oforganisms and the high level of skill in the area of genomics, thoseskilled in the art will readily be able to apply the teachings andguidance provided herein to essentially all other organisms. Forexample, the E. coli metabolic alterations exemplified herein canreadily be applied to other species by incorporating the same oranalogous encoding nucleic acid from species other than the referencedspecies. Such genetic alterations include, for example, geneticalterations of species homologs, in general, and in particular,orthologs, paralogs or nonorthologous gene displacements.

An ortholog is a gene or genes that are related by vertical descent andare responsible for substantially the same or identical functions indifferent organisms. For example, mouse epoxide hydrolase and humanepoxide hydrolase can be considered orthologs for the biologicalfunction of hydrolysis of epoxides. Genes are related by verticaldescent when, for example, they share sequence similarity of sufficientamount to indicate they are homologous, or related by evolution from acommon ancestor. Genes can also be considered orthologs if they sharethree-dimensional structure but not necessarily sequence similarity, ofa sufficient amount to indicate that they have evolved from a commonancestor to the extent that the primary sequence similarity is notidentifiable. Genes that are orthologous can encode proteins withsequence similarity of about 25% to 100% amino acid sequence identity.Genes encoding proteins sharing an amino acid similarity less that 25%can also be considered to have arisen by vertical descent if theirthree-dimensional structure also shows similarities. Members of theserine protease family of enzymes, including tissue plasminogenactivator and elastase, are considered to have arisen by verticaldescent from a common ancestor.

Orthologs include genes or their encoded gene products that through, forexample, evolution, have diverged in structure or overall activity. Forexample, where one species encodes a gene product exhibiting twofunctions and where such functions have been separated into distinctgenes in a second species, the three genes and their correspondingproducts are considered to be orthologs. For the production of abiochemical product, those skilled in the art will understand that theorthologous gene harboring the metabolic activity to be introduced ordisrupted is to be chosen for construction of the non-naturallyoccurring microorganism. An example of orthologs exhibiting separableactivities is where distinct activities have been separated intodistinct gene products between two or more species or within a singlespecies. A specific example is the separation of elastase proteolysisand plasminogen proteolysis, two types of serine protease activity, intodistinct molecules as plasminogen activator and elastase. A secondexample is the separation of mycoplasma 5′-3′ exonuclease and DrosophilaDNA polymerase III activity. The DNA polymerase from the first speciescan be considered an ortholog to either or both of the exonuclease orthe polymerase from the second species and vice versa.

In contrast, paralogs are homologs related by, for example, duplicationfollowed by evolutionary divergence and have similar or common, but notidentical functions. Paralogs can originate or derive from, for example,the same species or from a different species. For example, microsomalepoxide hydrolase (epoxide hydrolase I) and soluble epoxide hydrolase(epoxide hydrolase II) can be considered paralogs because they representtwo distinct enzymes, co-evolved from a common ancestor, that catalyzedistinct reactions and have distinct functions in the same species.Paralogs are proteins from the same species with significant sequencesimilarity to each other suggesting that they are homologous, or relatedthrough co-evolution from a common ancestor. Groups of paralogousprotein families include HipA homologs, luciferase genes, peptidases,and others.

A nonorthologous gene displacement is a nonorthologous gene from onespecies that can substitute for a referenced gene function in adifferent species. Substitution includes, for example, being able toperform substantially the same or a similar function in the species oforigin compared to the referenced function in the different species.Although generally, a nonorthologous gene displacement will beidentifiable as structurally related to a known gene encoding thereferenced function, less structurally related but functionally similargenes and their corresponding gene products nevertheless will still fallwithin the meaning of the term as it is used herein. Functionalsimilarity requires, for example, at least some structural similarity inthe active site or binding region of a nonorthologous gene productcompared to a gene encoding the function sought to be substituted.Therefore, a nonorthologous gene includes, for example, a paralog or anunrelated gene.

Therefore, in identifying and constructing the non-naturally occurringmicrobial organisms of the invention having 1,2-propanediol, n-propanol,1,3-propanediol or glycerol biosynthetic capability, those skilled inthe art will understand with applying the teaching and guidance providedherein to a particular species that the identification of metabolicmodifications can include identification and inclusion or inactivationof orthologs. To the extent that paralogs and/or nonorthologous genedisplacements are present in the referenced microorganism that encode anenzyme catalyzing a similar or substantially similar metabolic reaction,those skilled in the art also can utilize these evolutionally relatedgenes.

Orthologs, paralogs and nonorthologous gene displacements can bedetermined by methods well known to those skilled in the art. Forexample, inspection of nucleic acid or amino acid sequences for twopolypeptides will reveal sequence identity and similarities between thecompared sequences. Based on such similarities, one skilled in the artcan determine if the similarity is sufficiently high to indicate theproteins are related through evolution from a common ancestor.Algorithms well known to those skilled in the art, such as Align, BLAST,Clustal W and others compare and determine a raw sequence similarity oridentity, and also determine the presence or significance of gaps in thesequence which can be assigned a weight or score. Such algorithms alsoare known in the art and are similarly applicable for determiningnucleotide sequence similarity or identity. Parameters for sufficientsimilarity to determine relatedness are computed based on well knownmethods for calculating statistical similarity, or the chance of findinga similar match in a random polypeptide, and the significance of thematch determined. A computer comparison of two or more sequences can, ifdesired, also be optimized visually by those skilled in the art. Relatedgene products or proteins can be expected to have a high similarity, forexample, 25% to 100% sequence identity. Proteins that are unrelated canhave an identity which is essentially the same as would be expected tooccur by chance, if a database of sufficient size is scanned (about 5%).Sequences between 5% and 24% may or may not represent sufficienthomology to conclude that the compared sequences are related. Additionalstatistical analysis to determine the significance of such matches giventhe size of the data set can be carried out to determine the relevanceof these sequences.

Exemplary parameters for determining relatedness of two or moresequences using the BLAST algorithm, for example, can be as set forthbelow. Briefly, amino acid sequence alignments can be performed usingBLASTP version 2.0.8 (Jan. 5, 1999) and the following parameters:Matrix: 0 BLOSUM62; gap open: 11; gap extension: 1; x_dropoff: 50;expect: 10.0; wordsize: 3; filter: on. Nucleic acid sequence alignmentscan be performed using BLASTN version 2.0.6 (Sep. 16, 1998) and thefollowing parameters: Match: 1; mismatch: −2; gap open: 5; gapextension: 2; x_dropoff: 50; expect: 10.0; wordsize: 11; filter: off.Those skilled in the art will know what modifications can be made to theabove parameters to either increase or decrease the stringency of thecomparison, for example, and determine the relatedness of two or moresequences.

3.2 Microbial Organisms that Utilize Reducing Equivalents Produced bythe Metabolism of Methanol

Provided herein are methanol metabolic pathways engineered to improvethe availability of reducing equivalents, which can be used for theproduction of product molecules. Exemplary product molecules include,without limitation, 1,2-propanediol, n-propanol, 1,3-propanediol orglycerol, although given the teachings and guidance provided herein, itwill be recognized by one skilled in the art that any product moleculethat utilizes reducing equivalents in its production can exhibitenhanced production through the biosynthetic pathways provided herein.

Methanol is a relatively inexpensive organic feedstock that can bederived from synthesis gas components, CO and H₂, via catalysis.Methanol can be used as a source of reducing equivalents to increase themolar yield of product molecules from carbohydrates.

1,2-propanediol, n-propanol, 1,3-propanediol and/or glycerol arecompounds of commercial significance. Industrial uses of1,2-propanediol, n-propanol, 1,3-propanediol and/or glycerol include,for example, pharmaceutical formulations, humectants, solvents,sweeteners, preservative, food additives, monoglycerides, diglycerides,emulsifiers, antifreeze and de-icer agents, oil dispersants, solvents,resins, polyglycerol esters, moisturizer, oils, shortenings, margarines,and medical, personal care, cosmetic or pharmaceutical preparations.

There exists a need for the development of methods for effectivelyproducing commercial quantities of compounds, such as 1,2-propanedioland glycerol, as well as n-propanol and 1,3-propanediol.

Accordingly, provided herein is bioderived 1,2-propanediol producedaccording to the methods described herein and biobased productscomprising or obtained using the bioderived 1,2-propanediol. Thebiobased product can comprise at least 5%, at least 10%, at least 20%,at least 30%, at least 40% or at least 50% bioderived 1,2-propanediol.The biobased product can comprises a portion of said bioderived1,2-propanediol as a repeating unit. The biobased product can be amolded product obtained by molding the biobased product.

Also provided herein is bioderived glycerol produced according to themethods described herein and biobased products comprising or obtainedusing the bioderived glycerol. The biobased product can comprise atleast 5%, at least 10%, at least 20%, at least 30%, at least 40% or atleast 50% bioderived glycerol. The biobased product can comprises aportion of said bioderived glycerol as a repeating unit. The biobasedproduct can be a molded product obtained by molding the biobasedproduct.

Also provided herein is bioderived n-propanol produced according to themethods described herein and biobased products comprising or obtainedusing the bioderived n-propanol. The biobased product can comprise atleast 5%, at least 10%, at least 20%, at least 30%, at least 40% or atleast 50% bioderived n-propanol. The biobased product can comprises aportion of said bioderived n-propanol as a repeating unit. The biobasedproduct can be a molded product obtained by molding the biobasedproduct.

Also provided herein is bioderived 1,3-propanediol produced according tothe methods described herein and biobased products comprising orobtained using the bioderived 1,3-propanediol. The biobased product cancomprise at least 5%, at least 10%, at least 20%, at least 30%, at least40% or at least 50% bioderived 1,3-propanediol. The biobased product cancomprises a portion of said bioderived 1,3-propanediol as a repeatingunit. The biobased product can be a molded product obtained by moldingthe biobased product.

In numerous engineered pathways, realization of maximum product yieldsbased on carbohydrate feedstock is hampered by insufficient reducingequivalents or by loss of reducing equivalents to byproducts. Methanolis a relatively inexpensive organic feedstock that can be used togenerate reducing equivalents by employing one or more methanolmetabolic enzymes as shown in FIG. 1. The reducing equivalents producedby the metabolism of methanol by one or more of the methanol metabolicpathways can then be used to power the glucose to 1,2-propanediol,n-propanol, 1,3-propanediol and glycerol production pathways, forexample, as shown in FIGS. 2 and 3.

The product yields per C-mol of substrate of microbial cellssynthesizing reduced fermentation products such as 1,2-propanediol,n-propanol, 1,3-propanediol and glycerol are limited by insufficientreducing equivalents in the carbohydrate feedstock. Reducingequivalents, or electrons, can be extracted from methanol using one ormore of the enzymes described in FIG. 1. The reducing equivalents arethen passed to acceptors such as oxidized ferredoxins, oxidizedquinones, oxidized cytochromes, NAD(P)+, water, or hydrogen peroxide toform reduced ferredoxin, reduced quinones, reduced cytochromes, NAD(P)H,H₂, or water, respectively. Reduced ferredoxin, reduced quinones andNAD(P)H are particularly useful as they can serve as redox carriers forvarious Wood-Ljungdahl pathway, reductive TCA cycle, or product pathwayenzymes.

Specific examples of how additional redox availability from methanol canimprove the yield of reduced products such as 1,2-propanediol,n-propanol, 1,3-propanediol or glycerol are shown.

The maximum theoretical yield to produce n-propanol from glucose is 1.33moles n-propanol per mole of glucose under aerobic conditions via thepathways shown in FIG. 2. The reducing equivalents generated from themethanol metabolic pathways provided herein will be utilized to powerthe glucose to n-propanol production pathways. Theoretically, allcarbons in glucose will be conserved, thus resulting in a maximaltheoretical yield to produce n-propanol from glucose at 2 molesn-propanol per mole of glucose under either aerobic or anaerobicconditions as shown in FIG. 2:Carbohydrates: 1C₆H₁₂O₆→1.33C₃H₈O+2CO₂+0.67H₂OCarbohydrates+H₂: 1C₆H₁₂O₆+6H₂→2C₃H₈O+4H₂O

In a similar manner, the maximum theoretical yield of 1,2-propanediolcan be improved from 1.5 mol/mol to 2 mol/mol glucose. An exemplary fluxdistribution with the improved yields is shown in FIG. 2.Carbohydrates: 1C₆H₁₂O₆→1.5C₃H₈O₂+1.5CO₂Carbohydrates+H₂: 1C₆H₁₂O₆+4H₂→2C₃H₈O₂+2H₂O

Other exemplary products for which the yields on carbohydrates can beimproved by providing additional reducing equivalents are1,3-propanediol (1,3-PDO) and glycerol. 1,3-PDO is mainly used as abuilding block in the production of polymers. It can be formulated intoa variety of industrial products including composites, adhesives,laminates, coatings, moldings, aliphatic polyesters, copolyesters. It isalso a solvent and used as an antifreeze and wood paint. 1,3-PDO can bechemically synthesized via the hydration of acrolein or by thehydroformylation of ethylene oxide to afford 3-hydroxypropionaldehyde.The resulting aldehyde is hydrogenated to give 1,3-PDO. Additionally,1,3-PDO can be produced biologically (Nakamura and Whited, Curr Op Biol,14:454-9 (2003)); Mendes et al. Appl Microbiol Biotechnol [in press](2011)). The production of 1,3-PDO through fermentation of sugars has atheoretical yield of 1.5 mol 1,3-PDO per mol of glucose.2C₆H₁₂O₆→3C₃H₈O₂+3CO₂

When the combined feedstock strategy is applied to 1,3-PDO production,the reducing equivalents generated from the methanol metabolic pathwaysprovided herein can increase the 1,3-PDO theoretical yield based onglucose to 2 mol 1,3-PDO per mol of glucose by the pathways shown inFIG. 3.1C₆H₁₂O₆+4H₂→2C₃H₈O₂+2H₂O or1C₆H₁₂O₆+4CO+2H₂O→2C₃H₈O₂+4CO₂ o1C₆H₁₂O₆+2CO+2H₂→2C₃H₈O₂+2CO₂

Similarly, the production of glycerol through fermentation can beimproved by the combined feedstock strategy. The production of glycerolthrough fermentation has a theoretical yield of 1.71 mol glycerol permol of glucose.7C₆H₁₂O₆+6H₂O→12C₃H₈O₃+6CO₂

When the combined feedstocks strategy is applied to glycerol production,the reducing equivalents generated from the methanol metabolic pathwaysprovided herein can increase the glycerol theoretical yield from glucoseto 2 mol glycerol per mol of glucose with the pathways detailed in FIG.3.1C₆H₁₂O₆+2H₂→2C₃H₈O₃ or1C₆H₁₂O₆+2CO+2H₂O→2C₃H₈O₃+2CO₂ or1C₆H₁₂O₆+1CO+1H₂+1H₂O→2C₃H₈O₃+1CO₂

In a first aspect, provided herein is a non-naturally occurringmicrobial organism having a methanol metabolic pathway, wherein saidorganism comprises at least one exogenous nucleic acid encoding amethanol metabolic pathway enzyme expressed in a sufficient amount toenhance the availability of reducing equivalents in the presence ofmethanol. In certain embodiments, the methanol metabolic pathwaycomprises one or more enzymes selected from the group consisting of amethanol methyltransferase; a methylenetetrahydrofolate reductase; amethylenetetrahydrofolate dehydrogenase; a methenyltetrahydrofolatecyclohydrolase; a formyltetrahydrofolate deformylase; aformyltetrahydrofolate synthetase; a formate hydrogen lyase; ahydrogenase; a formate dehydrogenase; a methanol dehydrogenase; aformaldehyde activating enzyme; a formaldehyde dehydrogenase; aS-(hydroxymethyl)glutathione synthase; a glutathione-dependentformaldehyde dehydrogenase; and an S-formylglutathione hydrolase. Suchorganisms advantageously allow for the production of reducingequivalents, which can then be used by the organism for the productionof 1,2-propanediol, n-propanol, 1,3-propanediol or glycerol using anyone of the 1,2-propanediol, n-propanol, 1,3-propanediol or glycerolpathways provided herein.

In certain embodiments, the methanol metabolic pathway comprises 1A, 1B,1C, 1D, 1E, 1F, 1G, 1H, 1I, 1J, 1K, 1L, 1M, 1N, or 1O or any combinationof 1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 1I, 1J, 1K, 1L, 1M, 1N, and 1O,thereof, wherein 1A is a methanol methyltransferase; 1B is amethylenetetrahydrofolate reductase; 1C is a methylenetetrahydrofolatedehydrogenase; 1D is a methenyltetrahydrofolate cyclohydrolase; 1E is aformyltetrahydrofolate deformylase; 1F is a formyltetrahydrofolatesynthetase; 1G is a formate hydrogen lyase; 1H is a hydrogenase, 1I is aformate dehydrogenase; 1J is a methanol dehydrogenase; 1K is aformaldehyde activating enzyme; 1L is a formaldehyde dehydrogenase; 1Mis a S-(hydroxymethyl)glutathione synthase; 1N is glutathione-dependentformaldehyde dehydrogenase; and 1O is S-formylglutathione hydrolase. Insome embodiments, 1K is spontaneous. In other embodiments, 1K is aformaldehyde activating enzyme. In some embodiments, 1M is spontaneous.In other embodiments, 1M is a S-(hydroxymethyl)glutathione synthase.

In one embodiment, the methanol metabolic pathway comprises 1A. Inanother embodiment, the methanol metabolic pathway comprises 1B. Inanother embodiment, the methanol metabolic pathway comprises 1C. In yetanother embodiment, the methanol metabolic pathway comprises 1D. In oneembodiment, the methanol metabolic pathway comprises 1E. In anotherembodiment, the methanol metabolic pathway comprises 1F. In anotherembodiment, the methanol metabolic pathway comprises 1G. In yet anotherembodiment, the methanol metabolic pathway comprises 1H. In oneembodiment, the methanol metabolic pathway comprises 1I. In anotherembodiment, the methanol metabolic pathway comprises 1J. In anotherembodiment, the methanol metabolic pathway comprises 1K. In yet anotherembodiment, the methanol metabolic pathway comprises 1L. In yet anotherembodiment, the methanol metabolic pathway comprises 1M. In anotherembodiment, the methanol metabolic pathway comprises 1N. In yet anotherembodiment, the methanol metabolic pathway comprises 1O. Any combinationof two, three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen or fifteen methanol metabolic pathway enzymes 1A, 1B,1C, 1D, 1E, 1F, 1G, 1H, 1I, 1J, 1K, 1L, 1M, 1N, and 1O is alsocontemplated.

In some embodiments, the methanol metabolic pathway is a methanolmetabolic pathway depicted in FIG. 1.

In one aspect, provided herein is a non-naturally occurring microbialorganism having a methanol metabolic pathway, wherein said organismcomprises at least one exogenous nucleic acid encoding a methanolmetabolic pathway enzyme expressed in a sufficient amount to enhance theavailability of reducing equivalents in the presence of methanol,wherein said methanol metabolic pathway comprises: (i) 1A and 1B, (ii)1J; or (iii) 1J and 1K. In one embodiment, the methanol metabolicpathway comprises 1A and 1B. In another embodiment, the methanolmetabolic pathway comprises 1J. In one embodiment, the methanolmetabolic pathway comprises 1J and 1K. In certain embodiments, themethanol metabolic pathway comprises 1A, 1B, 1C, 1D, and 1E. In someembodiments. the methanol metabolic pathway comprises 1A, 1B, 1C, 1D and1F. In some embodiments, the methanol metabolic pathway comprises 1J,1C, 1D and 1E. In one embodiment, the methanol metabolic pathwaycomprises 1J, 1C, 1D and 1F. In another embodiment, the methanolmetabolic pathway comprises 1J and 1L. In yet another embodiment, themethanol metabolic pathway comprises 1J, 1M, 1N and 1O. In certainembodiments, the methanol metabolic pathway comprises 1J, 1N and 1O. Insome embodiments, the methanol metabolic pathway comprises 1J, 1K, 1C,1D and 1E. In one embodiment, the methanol metabolic pathway comprises1J, 1K, 1C, 1D and 1F. In some embodiments, 1K is spontaneous. In otherembodiments, 1K is a formaldehyde activating enzyme. In someembodiments, 1M is spontaneous. In other embodiments, 1M is aS-(hydroxymethyl)glutathione synthase.

In certain embodiments, the methanol metabolic pathway comprises 1I. Incertain embodiments, the methanol metabolic pathway comprises 1A, 1B,1C, 1D, 1E and 1I. In some embodiments, the methanol metabolic pathwaycomprises 1A, 1B, 1C, 1D, 1F and 1I. In some embodiments, the methanolmetabolic pathway comprises 1J, 1C, 1D, 1E and 1I. In one embodiment,the methanol metabolic pathway comprises 1J, 1C, 1D, 1F and 1I. Inanother embodiment, the methanol metabolic pathway comprises 1J, 1L and1I. In yet another embodiment, the methanol metabolic pathway comprises1J, 1M, 1N, 1O and 1I. In certain embodiments, the methanol metabolicpathway comprises 1J, 1N, 1O and 1I. In some embodiments, the methanolmetabolic pathway comprises 1J, 1K, 1C, 1D, 1E and 1I. In oneembodiment, the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1Fand 1I. In some embodiments, 1K is spontaneous. In other embodiments, 1Kis a formaldehyde activating enzyme. In some embodiments, 1M isspontaneous. In other embodiments, 1M is a S-(hydroxymethyl)glutathionesynthase.

In certain embodiments, the methanol metabolic pathway comprises 1G. Incertain embodiments, the methanol metabolic pathway comprises 1A, 1B,1C, 1D, 1E and 1G. In some embodiments. the methanol metabolic pathwaycomprises 1A, 1B, 1C, 1D, 1F and 1G. In some embodiments, the methanolmetabolic pathway comprises 1J, 1C, 1D, 1E and 1G. In one embodiment,the methanol metabolic pathway comprises 1J, 1C, 1D, 1F and 1G. Inanother embodiment, the methanol metabolic pathway comprises 1J, 1L and1G. In yet another embodiment, the methanol metabolic pathway comprises1J, 1M, 1N, 1O and 1G. In certain embodiments, the methanol metabolicpathway comprises 1J, 1N, 1O and 1G. In some embodiments, the methanolmetabolic pathway comprises 1J, 1K, 1C, 1D, 1E and 1G. In oneembodiment, the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1Fand 1G. In some embodiments, 1K is spontaneous. In other embodiments, 1Kis a formaldehyde activating enzyme. In some embodiments, 1M isspontaneous. In other embodiments, 1M is a S-(hydroxymethyl)glutathionesynthase.

In certain embodiments, the methanol metabolic pathway comprises 1G and1H In certain embodiments, the methanol metabolic pathway comprises 1A,1B, 1C, 1D, 1E, 1G and 1H. In some embodiments. the methanol metabolicpathway comprises 1A, 1B, 1C, 1D, 1F, 1G and 1H. In some embodiments,the methanol metabolic pathway comprises 1J, 1C, 1D, 1E, 1G and 1H. Inone embodiment, the methanol metabolic pathway comprises 1J, 1C, 1D, 1F,1G and 1H. In another embodiment, the methanol metabolic pathwaycomprises 1J, 1L, 1G and 1H. In yet another embodiment, the methanolmetabolic pathway comprises 1J, 1M, 1N, 1O, 1G and 1H. In certainembodiments, the methanol metabolic pathway comprises 1J, 1N, 1O, 1G and1H. In some embodiments, the methanol metabolic pathway comprises 1J,1K, 1C, 1D, 1E, 1G and 1H. In one embodiment, the methanol metabolicpathway comprises 1J, 1K, 1C, 1D, 1F, 1G and 1H. In some embodiments, 1Kis spontaneous. In other embodiments, 1K is a formaldehyde activatingenzyme. In some embodiments, 1M is spontaneous. In other embodiments, 1Mis a S-(hydroxymethyl)glutathione synthase.

In certain embodiments, the formation of 5-hydroxymethylglutathione fromformaldehyde is spontaneous (see, e.g., FIG. 1, step M). In someembodiments, the formation of 5-hydroxymethylglutathione fromformaldehyde is catalyzed by a S-(hydroxymethyl)glutathione synthase(see, e.g., FIG. 1, step M). In certain embodiments, the formation ofmethylene-THF from formaldehyde is spontaneous (see, e.g., FIG. 1, stepK). In certain embodiments, the formation of methylene-THF fromformaldehyde is catalyzed by a formaldehyde activating enzyme (see,e.g., FIG. 1, step K).

In certain embodiments, the organism comprises two, three, four, five,six or seven exogenous nucleic acids, each encoding a methanol metabolicpathway enzyme. In certain embodiments, the organism comprises twoexogenous nucleic acids, each encoding a methanol metabolic pathwayenzyme. In certain embodiments, the organism comprises three exogenousnucleic acids, each encoding a methanol metabolic pathway enzyme. Incertain embodiments, the organism comprises four exogenous nucleicacids, each encoding a methanol metabolic pathway enzyme. In certainembodiments, the organism comprises five exogenous nucleic acids, eachencoding a methanol metabolic pathway enzyme. In certain embodiments,the organism comprises six exogenous nucleic acids, each encoding amethanol metabolic pathway enzyme. In certain embodiments, the organismcomprises seven exogenous nucleic acids, each encoding a methanolmetabolic pathway enzyme.

Any non-naturally occurring eukaryotic organism comprising a methanolmetabolic pathway and engineered to comprise a methanol metabolicpathway enzyme, such as those provided herein, can be engineered tofurther comprise one or more 1,2-propanediol, n-propanol,1,3-propanediol or glycerol pathway enzymes.

In one embodiment, the non-naturally occurring microbial organismfurther comprises a 1,2-propanediol pathway, wherein said organismcomprises at least one exogenous nucleic acid encoding a 1,2-propanediolpathway enzyme expressed in a sufficient amount to produce1,2-propanediol. In certain embodiments, the 1,2-propanediol pathwayenzyme is selected from the group consisting of a methylglyoxalsynthase; a methylglyoxal reductase (acetol-forming); an acetolreductase; a methylglyoxal reductase (lactaldehyde-forming); and alactaldehyde reductase.

In another embodiment, the non-naturally occurring microbial organismfurther comprises a n-propanol pathway, wherein said organism comprisesat least one exogenous nucleic acid encoding a n-propanol pathway enzymeexpressed in a sufficient amount to produce n-propanol. In certainembodiments, the n-propanol pathway enzyme is selected from the groupconsisting of a methylglyoxal synthase; a methylglyoxal reductase(acetol-forming); an acetol reductase; a methylglyoxal reductase(lactaldehyde-forming); a lactaldehyde reductase; a 1,2-propanedioldehydratase; and a propanal reductase.

In one embodiment, the non-naturally occurring microbial organismfurther comprises a 1,3-propanediol pathway, wherein said organismcomprises at least one exogenous nucleic acid encoding a 1,3-propanediolpathway enzyme expressed in a sufficient amount to produce1,3-propanediol. In certain embodiments, the 1,3-propanediol pathwayenzyme is selected from the group consisting of aglyceraldehyde-3-phosphate reductase; a glycerol-3-phosphate phosphataseor a glycerol kinase; a glycerol dehydratase; a 3-hydroxypropanalreductase; a dihydroxyacetone phosphate phosphatase or adihydroxyacetone kinase; a dihydroxyacetone reductase; and adihydroxyacetone phosphate reductase.

In other embodiments, the non-naturally occurring microbial organism hasa glycerol pathway, wherein said organism comprises at least oneexogenous nucleic acid encoding a glycerol pathway enzyme expressed in asufficient amount to produce glycerol. In certain embodiments, theglycerol pathway enzyme is selected from the group consisting of aglyceraldehyde-3-phosphate reductase; a glycerol-3-phosphate phosphataseor a glycerol kinase; a dihydroxyacetone phosphate phosphatase or adihydroxyacetone kinase; a dihydroxyacetone reductase; and adihydroxyacetone phosphate reductase.

In some embodiments, the non-naturally occurring microbial organismshaving a 1,2-propanediol, n-propanol, 1,3-propanediol or glycerolpathway include a set of 1,2-propanediol, n-propanol, 1,3-propanediol orglycerol pathway enzymes.

Enzymes, genes and methods for engineering pathways from glycerol tovarious products, such as 1,2-propanediol, n-propanol, 1,3-propanediolor glycerol, into a microorganism, are now known in the art (see, e.g.,U.S. Publ. No. 2011/0201089, which is herein incorporated by referencein its entirety). A set of 1,2-propanediol, n-propanol, 1,3-propanediolor glycerol pathway enzymes represents a group of enzymes that canconvert glucose to 1,2-propanediol, n-propanol, 1,3-propanediol orglycerol, respectively, as shown in FIGS. 2 and 3. The additionalreducing equivalents obtained from the methanol metabolic pathways, asdisclosed herein, improve the yields of all these products whenutilizing carbohydrate-based feedstock.

Exemplary enzymes for the conversion of glucose to 1,2-propanediol(e.g., via methylglyoxyl) include a methylglyoxal synthase (FIG. 2, stepA); a methylglyoxal reductase (acetol-forming) (FIG. 2, step B); anacetol reductase (FIG. 2, step C); a methylglyoxal reductase(lactaldehyde-forming) (FIG. 2, step D); and a lactaldehyde reductase(FIG. 2, step E).

In one aspect, provided herein is a non-naturally occurring microbialorganism, comprising (1) a methanol metabolic pathway, wherein saidorganism comprises at least one exogenous nucleic acid encoding amethanol metabolic pathway enzyme in a sufficient amount to enhance theavailability of reducing equivalents in the presence of methanol; and(2) a 1,2-propanediol pathway, wherein said organism comprises at leastone exogenous nucleic acid encoding a 1,2-propanediol pathway enzymeexpressed in a sufficient amount to produce 1,2-propanediol. In oneembodiment, the at least one exogenous nucleic acid encoding themethanol metabolic pathway enzyme enhances the availability of reducingequivalents in the presence of methanol in a sufficient amount toincrease the amount of 1,2-propanediol produced by the non-naturallymicrobial organism. In some embodiments, the methanol metabolic pathwaycomprises any of the various combinations of methanol metabolic pathwayenzymes described above or elsewhere herein.

In certain embodiments, (1) the methanol metabolic pathway comprises:1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 1I, 1J, 1K, 1L, 1M, 1N, or 1O or anycombination of 1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 1I, 1J, 1K, 1L, 1M, 1N,or 1O, thereof, wherein 1A is a methanol methyltransferase; 1B is amethylenetetrahydrofolate reductase; 1C is a methylenetetrahydrofolatedehydrogenase; 1D is a methenyltetrahydrofolate cyclohydrolase; 1E is aformyltetrahydrofolate deformylase; 1F is a formyltetrahydrofolatesynthetase; 1G is a formate hydrogen lyase; 1H is a hydrogenase, 1I is aformate dehydrogenase; 1J is a methanol dehydrogenase; 1K is spontaneousor formaldehyde activating enzyme; 1L is a formaldehyde dehydrogenase;1M is spontaneous or a S-(hydroxymethyl)glutathione synthase; 1N isglutathione-dependent formaldehyde dehydrogenase and 1O isS-formylglutathione hydrolase; and (2) the 1,2-propanediol pathwaycomprises 2A, 2B, 2C, 2D or 2E, or any combination thereof, wherein 2Ais a methylglyoxal synthase; 2B is a methylglyoxal reductase(acetol-forming); 2C is an acetol reductase; 2D is a methylglyoxalreductase (lactaldehyde-forming); and 2E is a lactaldehyde reductase. Insome embodiments, 1K is spontaneous. In other embodiments, 1K is aformaldehyde activating enzyme. In some embodiments, 1M is spontaneous.In other embodiments, 1M is a S-(hydroxymethyl)glutathione synthase.

In one embodiment, the 1,2-propanediol pathway comprises 2A. In anotherembodiment, the 1,2-propanediol pathway comprises 2B. In an embodiment,the 1,2-propanediol pathway comprises 2C. In another embodiment, the1,2-propanediol pathway comprises 2D. In another embodiment, the1,2-propanediol pathway comprises 2E. Any combination of two, three,four or five 1,2-propanediol pathway enzymes 2A, 2B, 2C, 2D and 2E isalso contemplated.

In some embodiments, the methanol metabolic pathway is a methanolmetabolic pathway depicted in FIG. 1, and the 1,2-propanediol pathway isa 1,2-propanediol pathway depicted in FIG. 2.

Exemplary sets of 1,2-propanediol pathway enzymes to convert glucose to1,2-propanediol (e.g., via dicydroxyacetone phosphate and methylglyoxal)according to FIG. 2 include (i) 2A, 2B and 2C; (ii) 2A, 2D and 2E; and(iii) 2A, 2D and 2E.

In one embodiment, (1) the methanol metabolic pathway comprises 1A and1B; and (2) the 1,2-propanediol pathway comprises 2A, 2B and 2C. Inanother embodiment, (1) the methanol metabolic pathway comprises 1J; and(2) the 1,2-propanediol pathway comprises 2A, 2B and 2C. In oneembodiment, (1) the methanol metabolic pathway comprises 1J and 1K; and(2) the 1,2-propanediol pathway comprises 2A, 2B and 2C. In certainembodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C,1D, and 1E; and (2) the 1,2-propanediol pathway comprises 2A, 2B and 2C.In some embodiments (1) the methanol metabolic pathway comprises 1A, 1B,1C, 1D and 1F; and (2) the 1,2-propanediol pathway comprises 2A, 2B and2C. In some embodiments, (1) the methanol metabolic pathway comprises1J, 1C, 1D and 1E; and (2) the 1,2-propanediol pathway comprises 2A, 2Band 2C. In one embodiment, (1) the methanol metabolic pathway comprises1J, 1C, 1D and 1F; and (2) the 1,2-propanediol pathway comprises 2A, 2Band 2C. In another embodiment, (1) the methanol metabolic pathwaycomprises 1J and 1L; and (2) the 1,2-propanediol pathway comprises 2A,2B and 2C. In yet another embodiment, (1) the methanol metabolic pathwaycomprises 1J, 1M, 1N and 1O; and (2) the 1,2-propanediol pathwaycomprises 2A, 2B and 2C. In certain embodiments, (1) the methanolmetabolic pathway comprises 1J, 1N and 1O; and (2) the 1,2-propanediolpathway comprises 2A, 2B and 2C. In some embodiments, (1) the methanolmetabolic pathway comprises 1J, 1K, 1C, 1D and 1E; and (2) the1,2-propanediol pathway comprises 2A, 2B and 2C. In one embodiment, (1)the methanol metabolic pathway comprises 1J, 1K, 1C, 1D and 1F; and (2)the 1,2-propanediol pathway comprises 2A, 2B and 2C. In certainembodiments, (1) the methanol metabolic pathway comprises 1I; and (2)the 1,2-propanediol pathway comprises 2A, 2B and 2C. In certainembodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C,1D, 1E and 1I; and (2) the 1,2-propanediol pathway comprises 2A, 2B and2C. In some embodiments, (1) the methanol metabolic pathway comprises1A, 1B, 1C, 1D, 1F and 1I; and (2) the 1,2-propanediol pathway comprises2A, 2B and 2C. In some embodiments, (1) the methanol metabolic pathwaycomprises 1J, 1C, 1D, 1E and 1I; and (2) the 1,2-propanediol pathwaycomprises 2A, 2B and 2C. In one embodiment, (1) the methanol metabolicpathway comprises 1J, 1C, 1D, 1F and 1I; and (2) the 1,2-propanediolpathway comprises 2A, 2B and 2C. In another embodiment, (1) the methanolmetabolic pathway comprises 1J, 1L and 1I; and (2) the 1,2-propanediolpathway comprises 2A, 2B and 2C. In yet another embodiment, (1) themethanol metabolic pathway comprises 1J, 1M, 1N, 1O and 1I; and (2) the1,2-propanediol pathway comprises 2A, 2B and 2C. In certain embodiments,(1) the methanol metabolic pathway comprises 1J, 1N, 1O and 1I; and (2)the 1,2-propanediol pathway comprises 2A, 2B and 2C. In someembodiments, (1) the methanol metabolic pathway comprises 1J, 1K, 1C,1D, 1E and 1I; and (2) the 1,2-propanediol pathway comprises 2A, 2B and2C. In one embodiment, (1) the methanol metabolic pathway comprises 1J,1K, 1C, 1D, 1F and 1I; and (2) the 1,2-propanediol pathway comprises 2A,2B and 2C. In certain embodiments, (1) the methanol metabolic pathwaycomprises 1G; and (2) the 1,2-propanediol pathway comprises 2A, 2B and2C. In certain embodiments, (1) the methanol metabolic pathway comprises1A, 1B, 1C, 1D, 1E and 1G; and (2) the 1,2-propanediol pathway comprises2A, 2B and 2C. In some embodiments, (1) the methanol metabolic pathwaycomprises 1A, 1B, 1C, 1D, 1F and 1G; and (2) the 1,2-propanediol pathwaycomprises 2A, 2B and 2C. In some embodiments, (1) the methanol metabolicpathway comprises 1J, 1C, 1D, 1E and 1G; and (2) the 1,2-propanediolpathway comprises 2A, 2B and 2C. In one embodiment, (1) the methanolmetabolic pathway comprises 1J, 1C, 1D, 1F and 1G; and (2) the1,2-propanediol pathway comprises 2A, 2B and 2C. In another embodiment,(1) the methanol metabolic pathway comprises 1J, 1L and 1G; and (2) the1,2-propanediol pathway comprises 2A, 2B and 2C. In yet anotherembodiment, (1) the methanol metabolic pathway comprises 1J, 1M, 1N, 1Oand 1G; and (2) the 1,2-propanediol pathway comprises 2A, 2B and 2C. Incertain embodiments, (1) the methanol metabolic pathway comprises 1J,1N, 1O and 1G; and (2) the 1,2-propanediol pathway comprises 2A, 2B and2C. In some embodiments, (1) the methanol metabolic pathway comprises1J, 1K, 1C, 1D, 1E and 1G; and (2) the 1,2-propanediol pathway comprises2A, 2B and 2C. In one embodiment, (1) the methanol metabolic pathwaycomprises 1J, 1K, 1C, 1D, 1F and 1G; and (2) the 1,2-propanediol pathwaycomprises 2A, 2B and 2C. In certain embodiments, (1) the methanolmetabolic pathway comprises 1G and 1H; and (2) the 1,2-propanediolpathway comprises 2A, 2B and 2C. In certain embodiments, (1) themethanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1E, 1G and 1H; and(2) the 1,2-propanediol pathway comprises 2A, 2B and 2C. In someembodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C,1D, 1F, 1G and 1H; and (2) the 1,2-propanediol pathway comprises 2A, 2Band 2C. In some embodiments, (1) the methanol metabolic pathwaycomprises 1J, 1C, 1D, 1E, 1G and 1H; and (2) the 1,2-propanediol pathwaycomprises 2A, 2B and 2C. In one embodiment, (1) the methanol metabolicpathway comprises 1J, 1C, 1D, 1F, 1G and 1H; and (2) the 1,2-propanediolpathway comprises 2A, 2B and 2C. In another embodiment, (1) the methanolmetabolic pathway comprises 1J, 1L, 1G and 1H; and (2) the1,2-propanediol pathway comprises 2A, 2B and 2C. In yet anotherembodiment, (1) the methanol metabolic pathway comprises 1J, 1M, 1N, 1O,1G and 1H; and (2) the 1,2-propanediol pathway comprises 2A, 2B and 2C.In certain embodiments, (1) the methanol metabolic pathway comprises 1J,1N, 1O, 1G and 1H; and (2) the 1,2-propanediol pathway comprises 2A, 2Band 2C. In some embodiments, (1) the methanol metabolic pathwaycomprises 1J, 1K, 1C, 1D, 1E, 1G and 1H; and (2) the 1,2-propanediolpathway comprises 2A, 2B and 2C. In one embodiment, (1) the methanolmetabolic pathway comprises 1J, 1K, 1C, 1D, 1F, 1G and 1H; and (2) the1,2-propanediol pathway comprises 2A, 2B and 2C. In some embodiments, 1Kis spontaneous. In other embodiments, 1K is a formaldehyde activatingenzyme. In some embodiments, 1M is spontaneous.

In one embodiment, (1) the methanol metabolic pathway comprises 1A and1B; and (2) the 1,2-propanediol pathway comprises 2A, 2D and 2E. Inanother embodiment, (1) the methanol metabolic pathway comprises 1J; and(2) the 1,2-propanediol pathway comprises 2A, 2D and 2E. In oneembodiment, (1) the methanol metabolic pathway comprises 1J and 1K; and(2) the 1,2-propanediol pathway comprises 2A, 2D and 2E. In certainembodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C,1D, and 1E; and (2) the 1,2-propanediol pathway comprises 2A, 2D and 2E.In some embodiments, (1) the methanol metabolic pathway comprises 1A,1B, 1C, 1D and 1F; and (2) the 1,2-propanediol pathway comprises 2A, 2Dand 2E. In some embodiments, (1) the methanol metabolic pathwaycomprises 1J, 1C, 1D and 1E; and (2) the 1,2-propanediol pathwaycomprises 2A, 2D and 2E. In one embodiment, (1) the methanol metabolicpathway comprises 1J, 1C, 1D and 1F; and (2) the 1,2-propanediol pathwaycomprises 2A, 2D and 2E. In another embodiment, (1) the methanolmetabolic pathway comprises 1J and 1L; and (2) the 1,2-propanediolpathway comprises 2A, 2D and 2E. In yet another embodiment, (1) themethanol metabolic pathway comprises 1J, 1M, 1N and 1O; and (2) the1,2-propanediol pathway comprises 2A, 2D and 2E. In certain embodiments,(1) the methanol metabolic pathway comprises 1J, 1N and 1O; and (2) the1,2-propanediol pathway comprises 2A, 2D and 2E. In some embodiments,(1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D and 1E; and(2) the 1,2-propanediol pathway comprises 2A, 2D and 2E. In oneembodiment, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1Dand 1F; and (2) the 1,2-propanediol pathway comprises 2A, 2D and 2E. Incertain embodiments, (1) the methanol metabolic pathway comprises 1I;and (2) the 1,2-propanediol pathway comprises 2A, 2D and 2E. In certainembodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C,1D, 1E and 1I; and (2) the 1,2-propanediol pathway comprises 2A, 2D and2E. In some embodiments, (1) the methanol metabolic pathway comprises1A, 1B, 1C, 1D, 1F and 1I; and (2) the 1,2-propanediol pathway comprises2A, 2D and 2E. In some embodiments, (1) the methanol metabolic pathwaycomprises 1J, 1C, 1D, 1E and 1I; and (2) the 1,2-propanediol pathwaycomprises 2A, 2D and 2E. In one embodiment, (1) the methanol metabolicpathway comprises 1J, 1C, 1D, 1F and 1I; and (2) the 1,2-propanediolpathway comprises 2A, 2D and 2E. In another embodiment, (1) the methanolmetabolic pathway comprises 1J, 1L and 1I; and (2) the 1,2-propanediolpathway comprises 2A, 2D and 2E. In yet another embodiment, (1) themethanol metabolic pathway comprises 1J, 1M, 1N, 1O and 1I; and (2) the1,2-propanediol pathway comprises 2A, 2D and 2E. In certain embodiments,(1) the methanol metabolic pathway comprises 1J, 1N, 1O and 1I; and (2)the 1,2-propanediol pathway comprises 2A, 2D and 2E. In someembodiments, (1) the methanol metabolic pathway comprises 1J, 1K, 1C,1D, 1E and 1I; and (2) the 1,2-propanediol pathway comprises 2A, 2D and2E. In one embodiment, (1) the methanol metabolic pathway comprises 1J,1K, 1C, 1D, 1F and 1I; and (2) the 1,2-propanediol pathway comprises 2A,2D and 2E. In certain embodiments, (1) the methanol metabolic pathwaycomprises 1G; and (2) the 1,2-propanediol pathway comprises 2A, 2D and2E. In certain embodiments, (1) the methanol metabolic pathway comprises1A, 1B, 1C, 1D, 1E and 1G; and (2) the 1,2-propanediol pathway comprises2A, 2D and 2E. In some embodiments, (1) the methanol metabolic pathwaycomprises 1A, 1B, 1C, 1D, 1F and 1G; and (2) the 1,2-propanediol pathwaycomprises 2A, 2D and 2E. In some embodiments, (1) the methanol metabolicpathway comprises 1J, 1C, 1D, 1E and 1G; and (2) the 1,2-propanediolpathway comprises 2A, 2D and 2E. In one embodiment, (1) the methanolmetabolic pathway comprises 1J, 1C, 1D, 1F and 1G; and (2) the1,2-propanediol pathway comprises 2A, 2D and 2E. In another embodiment,(1) the methanol metabolic pathway comprises 1J, 1L and 1G; and (2) the1,2-propanediol pathway comprises 2A, 2D and 2E. In yet anotherembodiment, (1) the methanol metabolic pathway comprises 1J, 1M, 1N, 1Oand 1G; and (2) the 1,2-propanediol pathway comprises 2A, 2D and 2E. Incertain embodiments, (1) the methanol metabolic pathway comprises 1J,1N, 1O and 1G; and (2) the 1,2-propanediol pathway comprises 2A, 2D and2E. In some embodiments, (1) the methanol metabolic pathway comprises1J, 1K, 1C, 1D, 1E and 1G; and (2) the 1,2-propanediol pathway comprises2A, 2D and 2E. In one embodiment, (1) the methanol metabolic pathwaycomprises 1J, 1K, 1C, 1D, 1F and 1G; and (2) the 1,2-propanediol pathwaycomprises 2A, 2D and 2E. In certain embodiments, (1) the methanolmetabolic pathway comprises 1G and 1H; and (2) the 1,2-propanediolpathway comprises 2A, 2D and 2E. In certain embodiments, (1) themethanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1E, 1G and 1H; and(2) the 1,2-propanediol pathway comprises 2A, 2D and 2E. In someembodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C,1D, 1F, 1G and 1H; and (2) the 1,2-propanediol pathway comprises 2A, 2Dand 2E. In some embodiments, (1) the methanol metabolic pathwaycomprises 1J, 1C, 1D, 1E, 1G and 1H; and (2) the 1,2-propanediol pathwaycomprises 2A, 2D and 2E. In one embodiment, (1) the methanol metabolicpathway comprises 1J, 1C, 1D, 1F, 1G and 1H; and (2) the 1,2-propanediolpathway comprises 2A, 2D and 2E. In another embodiment, (1) the methanolmetabolic pathway comprises 1J, 1L, 1G and 1H; and (2) the1,2-propanediol pathway comprises 2A, 2D and 2E. In yet anotherembodiment, (1) the methanol metabolic pathway comprises 1J, 1M, 1N, 1O,1G and 1H; and (2) the 1,2-propanediol pathway comprises 2A, 2D and 2E.In certain embodiments, (1) the methanol metabolic pathway comprises 1J,1N, 1O, 1G and 1H; and (2) the 1,2-propanediol pathway comprises 2A, 2Dand 2E. In some embodiments, (1) the methanol metabolic pathwaycomprises 1J, 1K, 1C, 1D, 1E, 1G and 1H; and (2) the 1,2-propanediolpathway comprises 2A, 2D and 2E. In one embodiment, (1) the methanolmetabolic pathway comprises 1J, 1K, 1C, 1D, 1F, 1G and 1H; and (2) the1,2-propanediol pathway comprises 2A, 2D and 2E. In some embodiments, 1Kis spontaneous. In other embodiments, 1K is a formaldehyde activatingenzyme. In some embodiments, 1M is spontaneous.

In one embodiment, (1) the methanol metabolic pathway comprises 1A and1B; and (2) the 1,2-propanediol pathway comprises 2A, 2D and 2E. Inanother embodiment, (1) the methanol metabolic pathway comprises 1J; and(2) the 1,2-propanediol pathway comprises 2A, 2D and 2E. In oneembodiment, (1) the methanol metabolic pathway comprises 1J and 1K; and(2) the 1,2-propanediol pathway comprises 2A, 2D and 2E. In certainembodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C,1D, and 1E; and (2) the 1,2-propanediol pathway comprises 2A, 2D and 2E.In some embodiments, (1) the methanol metabolic pathway comprises 1A,1B, 1C, 1D and 1F; and (2) the 1,2-propanediol pathway comprises 2A, 2Dand 2E. In some embodiments, (1) the methanol metabolic pathwaycomprises 1J, 1C, 1D and 1E; and (2) the 1,2-propanediol pathwaycomprises 2A, 2D and 2E. In one embodiment, (1) the methanol metabolicpathway comprises 1J, 1C, 1D and 1F; and (2) the 1,2-propanediol pathwaycomprises 2A, 2D and 2E. In another embodiment, (1) the methanolmetabolic pathway comprises 1J and 1L; and (2) the 1,2-propanediolpathway comprises 2A, 2D and 2E. In yet another embodiment, (1) themethanol metabolic pathway comprises 1J, 1M, 1N and 1O; and (2) the1,2-propanediol pathway comprises 2A, 2D and 2E. In certain embodiments,(1) the methanol metabolic pathway comprises 1J, 1N and 1O; and (2) the1,2-propanediol pathway comprises 2A, 2D and 2E. In some embodiments,(1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D and 1E; and(2) the 1,2-propanediol pathway comprises 2A, 2D and 2E. In oneembodiment, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1Dand 1F; and (2) the 1,2-propanediol pathway comprises 2A, 2D and 2E. Incertain embodiments, (1) the methanol metabolic pathway comprises 1I;and (2) the 1,2-propanediol pathway comprises 2A, 2D and 2E. In certainembodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C,1D, 1E and 1I; and (2) the 1,2-propanediol pathway comprises 2A, 2D and2E. In some embodiments, (1) the methanol metabolic pathway comprises1A, 1B, 1C, 1D, 1F and 1I; and (2) the 1,2-propanediol pathway comprises2A, 2D and 2E. In some embodiments, (1) the methanol metabolic pathwaycomprises 1J, 1C, 1D, 1E and 1I; and (2) the 1,2-propanediol pathwaycomprises 2A, 2D and 2E. In one embodiment, (1) the methanol metabolicpathway comprises 1J, 1C, 1D, 1F and 1I; and (2) the 1,2-propanediolpathway comprises 2A, 2D and 2E. In another embodiment, (1) the methanolmetabolic pathway comprises 1J, 1L and 1I; and (2) the 1,2-propanediolpathway comprises 2A, 2D and 2E. In yet another embodiment, (1) themethanol metabolic pathway comprises 1J, 1M, 1N, 1O and 1I; and (2) the1,2-propanediol pathway comprises 2A, 2D and 2E. In certain embodiments,(1) the methanol metabolic pathway comprises 1J, 1N, 1O and 1I; and (2)the 1,2-propanediol pathway comprises 2A, 2D and 2E. In someembodiments, (1) the methanol metabolic pathway comprises 1J, 1K, 1C,1D, 1E and 1I; and (2) the 1,2-propanediol pathway comprises 2A, 2D and2E. In one embodiment, (1) the methanol metabolic pathway comprises 1J,1K, 1C, 1D, 1F and 1I; and (2) the 1,2-propanediol pathway comprises 2A,2D and 2E. In certain embodiments, (1) the methanol metabolic pathwaycomprises 1G; and (2) the 1,2-propanediol pathway comprises 2A, 2D and2E. In certain embodiments, (1) the methanol metabolic pathway comprises1A, 1B, 1C, 1D, 1E and 1G; and (2) the 1,2-propanediol pathway comprises2A, 2D and 2E. In some embodiments, (1) the methanol meta bolic pathwaycomprises 1A, 1B, 1C, 1D, 1F and 1G; and (2) the 1,2-propanediol pathwaycomprises 2A, 2D and 2E. In some embodiments, (1) the methanol metabolicpathway comprises 1J, 1C, 1D, 1E and 1G; and (2) the 1,2-propanediolpathway comprises 2A, 2D and 2E. In one embodiment, (1) the methanolmetabolic pathway comprises 1J, 1C, 1D, 1F and 1G; and (2) the1,2-propanediol pathway comprises 2A, 2D and 2E. In another embodiment,(1) the methanol metabolic pathway comprises 1J, 1L and 1G; and (2) the1,2-propanediol pathway comprises 2A, 2D and 2E. In yet anotherembodiment, (1) the methanol metabolic pathway comprises 1J, 1M, 1N, 1Oand 1G; and (2) the 1,2-propanediol pathway comprises 2A, 2D and 2E. Incertain embodiments, (1) the methanol metabolic pathway comprises 1J,1N, 1O and 1G; and (2) the 1,2-propanediol pathway comprises 2A, 2D and2E. In some embodiments, (1) the methanol metabolic pathway comprises1J, 1K, 1C, 1D, 1E and 1G; and (2) the 1,2-propanediol pathway comprises2A, 2D and 2E. In one embodiment, (1) the methanol metabolic pathwaycomprises 1J, 1K, 1C, 1D, 1F and 1G; and (2) the 1,2-propanediol pathwaycomprises 2A, 2D and 2E. In certain embodiments, (1) the methanolmetabolic pathway comprises 1G and 1H; and (2) the 1,2-propanediolpathway comprises 2A, 2D and 2E. In certain embodiments, (1) themethanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1E, 1G and 1H; and(2) the 1,2-propanediol pathway comprises 2A, 2D and 2E. In someembodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C,1D, 1F, 1G and 1H; and (2) the 1,2-propanediol pathway comprises 2A, 2Dand 2E. In some embodiments, (1) the methanol metabolic pathwaycomprises 1J, 1C, 1D, 1E, 1G and 1H; and (2) the 1,2-propanediol pathwaycomprises 2A, 2D and 2E. In one embodiment, (1) the methanol metabolicpathway comprises 1J, 1C, 1D, 1F, 1G and 1H; and (2) the 1,2-propanediolpathway comprises 2A, 2D and 2E. In another embodiment, (1) the methanolmetabolic pathway comprises 1J, 1L, 1G and 1H; and (2) the1,2-propanediol pathway comprises 2A, 2D and 2E. In yet anotherembodiment, (1) the methanol metabolic pathway comprises 1J, 1M, 1N, 1O,1G and 1H; and (2) the 1,2-propanediol pathway comprises 2A, 2D and 2E.In certain embodiments, (1) the methanol metabolic pathway comprises 1J,1N, 1O, 1G and 1H; and (2) the 1,2-propanediol pathway comprises 2A, 2Dand 2E. In some embodiments, (1) the methanol metabolic pathwaycomprises 1J, 1K, 1C, 1D, 1E, 1G and 1H; and (2) the 1,2-propanediolpathway comprises 2A, 2D and 2E. In one embodiment, (1) the methanolmetabolic pathway comprises 1J, 1K, 1C, 1D, 1F, 1G and 1H; and (2) the1,2-propanediol pathway comprises 2A, 2D and 2E. In some embodiments, 1Kis spontaneous. In other embodiments, 1K is a formaldehyde activatingenzyme. In some embodiments, 1M is spontaneous.

In one embodiment, the non-naturally occurring microbial organismcomprises (1) a methanol metabolic pathway comprising 1A and 1B; 1J; 1Jand 1K; 1A, 1B, 1C, 1D, and 1E; 1A, 1B, 1C, 1D and 1F; 1J, 1C, 1D and1E; 1J, 1C, 1D and 1F; 1J and 1L; 1J, 1M, 1N and 1O; 1J, 1N and 1O; 1J,1K, 1C, 1D and 1E; 1J, 1K, 1C, 1D and 1F; 1I; 1A, 1B, 1C, 1D, 1E and 1I;1A, 1B, 1C, 1D, 1F and 1I; 1J, 1C, 1D, 1E and 1I; 1J, 1C, 1D, 1F and 1I;1J, 1L and 1I; 1J, 1M, 1N, 1O and 1I; 1J, 1N, 1O and 1I; 1J, 1K, 1C, 1D,1E and 1I; 1J, 1K, 1C, 1D, 1F and 1I; 1G; 1A, 1B, 1C, 1D, 1E and 1G; 1A,1B, 1C, 1D, 1F and 1G; 1J, 1C, 1D, 1E and 1G; 1J, 1C, 1D, 1F and 1G; 1J,1L and 1G; 1J, 1M, 1N, 1O and 1G; 1J, 1N, 1O and 1G; 1J, 1K, 1C, 1D, 1Eand 1G; 1J, 1K, 1C, 1D, 1F and 1G; 1G and 1H; 1A, 1B, 1C, 1D, 1E, 1G and1H; 1A, 1B, 1C, 1D, 1F, 1G and 1H; 1J, 1C, 1D, 1E, 1G and 1H; 1J, 1C,1D, 1F, 1G and 1H; 1J, 1L, 1G and 1H; 1J, 1M, 1N, 1O, 1G and 1H; 1J, 1N,1O, 1G and 1H; 1J, 1K, 1C, 1D, 1E, 1G and 1H; or 1J, 1K, 1C, 1D, 1F, 1Gand 1H; and (2) a 1,2-propanediol pathway. In some embodiments, 1K isspontaneous. In other embodiments, 1K is a formaldehyde activatingenzyme. In some embodiments, 1M is spontaneous. In other embodiments, 1Mis a S-(hydroxymethyl)glutathione synthase.

Any methanol metabolic pathway provided herein can be combined with any1,2-propanediol pathway provided herein.

Exemplary enzymes for the conversion of glucose to n-propanol (e.g., viamethylglyoxyl) include a methylglyoxal synthase (FIG. 2, step A); amethylglyoxal reductase (acetol-forming) (FIG. 2, step B); an acetolreductase (FIG. 2, step C); a methylglyoxal reductase(lactaldehyde-forming) (FIG. 2, step D); a lactaldehyde reductase (FIG.2, step E); a 1,2-propanediol dehydratase (FIG. 2, step F); and apropanal reductase (FIG. 2, step G).

In another aspect, provided herein is a non-naturally occurringmicrobial organism, comprising (1) a methanol metabolic pathway, whereinsaid organism comprises at least one exogenous nucleic acid encoding amethanol metabolic pathway enzyme in a sufficient amount to enhance theavailability of reducing equivalents in the presence of methanol; and(2) an n-propanol pathway, wherein said organism comprises at least oneexogenous nucleic acid encoding an n-propanol pathway enzyme expressedin a sufficient amount to produce n-propanol. In one embodiment, the atleast one exogenous nucleic acid encoding the methanol metabolic pathwayenzyme enhances the availability of reducing equivalents in the presenceof methanol in a sufficient amount to increase the amount of n-propanolproduced by the non-naturally microbial organism. In some embodiments,the methanol metabolic pathway comprises any of the various combinationsof methanol metabolic pathway enzymes described above or elsewhereherein.

In certain embodiments, (1) the methanol metabolic pathway comprises:1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 1I, 1J, 1K, 1L, 1M, 1N, or 1O or anycombination of 1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 1I, 1J, 1K, 1L, 1M, 1N,or 1O, thereof, wherein 1A is a methanol methyltransferase; 1B is amethylenetetrahydrofolate reductase; 1C is a methylenetetrahydrofolatedehydrogenase; 1D is a methenyltetrahydrofolate cyclohydrolase; 1E is aformyltetrahydrofolate deformylase; 1F is a formyltetrahydrofolatesynthetase; 1G is a formate hydrogen lyase; 1H is a hydrogenase, 1I is aformate dehydrogenase; 1J is a methanol dehydrogenase; 1K is spontaneousor formaldehyde activating enzyme; 1L is a formaldehyde dehydrogenase;1M is spontaneous or a S-(hydroxymethyl)glutathione synthase; 1N isglutathione-dependent formaldehyde dehydrogenase and 1O isS-formylglutathione hydrolase; and (2) the n-propanol pathway comprises2A, 2B, 2C, 2D, 2E, 2F or 2G or any combination thereof, wherein 2A is amethylglyoxal synthase; 2B is a methylglyoxal reductase(acetol-forming); 2C is an acetol reductase; 2D is a methylglyoxalreductase (lactaldehyde-forming); 2E is a lactaldehyde reductase; 2F isa 1,2-propanediol dehydratase; and 2G is a propanal reductase. In someembodiments, 1K is spontaneous. In other embodiments, 1K is aformaldehyde activating enzyme. In some embodiments, 1M is spontaneous.In other embodiments, 1M is a S-(hydroxymethyl)glutathione synthase.

In one embodiment, the n-propanol pathway comprises 2A. In anotherembodiment, the n-propanol pathway comprises 2B. In an embodiment, then-propanol pathway comprises 2C. In another embodiment, the n-propanolpathway comprises 2D. In one embodiment, the n-propanol pathwaycomprises 2E. In yet another embodiment, the n-propanol pathwaycomprises 2F. In one embodiment, the n-propanol pathway comprises 2G.Any combination of two, three, four, five, six or seven n-propanolpathway enzymes 2A, 2B, 2C, 2D, 2E, 2F and 2G is also contemplated.

In some embodiments, the methanol metabolic pathway is a methanolmetabolic pathway depicted in FIG. 1, and the n-propanol pathway is ann-propanol pathway depicted in FIG. 2.

Exemplary sets of n-propanol pathway enzymes to convert to convertglucose to n-propanol (e.g., via dicydroxyacetone phosphate andmethylglyoxal) according to FIG. 2 include (i) 2A, 2B, 2C, 2F and 2G;and (ii) 2A, 2D, 2E, 2F and 2G.

In one embodiment, (1) the methanol metabolic pathway comprises 1A and1B; and (2) the n-propanol pathway comprises 2A, 2B, 2C, 2F and 2G. Inanother embodiment, (1) the methanol metabolic pathway comprises 1J; and(2) the n-propanol pathway comprises 2A, 2B, 2C, 2F and 2G. In oneembodiment, (1) the methanol metabolic pathway comprises 1J and 1K; and(2) the n-propanol pathway comprises 2A, 2B, 2C, 2F and 2G. In certainembodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C,1D, and 1E; and (2) the n-propanol pathway comprises 2A, 2B, 2C, 2F and2G. In some embodiments, (1) the methanol metabolic pathway comprises1A, 1B, 1C, 1D and 1F; and (2) the n-propanol pathway comprises 2A, 2B,2C, 2F and 2G. In some embodiments, (1) the methanol metabolic pathwaycomprises 1J, 1C, 1D and 1E; and (2) the n-propanol pathway comprises2A, 2B, 2C, 2F and 2G. In one embodiment, (1) the methanol metabolicpathway comprises 1J, 1C, 1D and 1F; and (2) the n-propanol pathwaycomprises 2A, 2B, 2C, 2F and 2G. In another embodiment, (1) the methanolmetabolic pathway comprises 1J and 1L; and (2) the n-propanol pathwaycomprises 2A, 2B, 2C, 2F and 2G. In yet another embodiment, (1) themethanol metabolic pathway comprises 1J, 1M, 1N and 1O; and (2) then-propanol pathway comprises 2A, 2B, 2C, 2F and 2G. In certainembodiments, (1) the methanol metabolic pathway comprises 1J, 1N and 1O;and (2) the n-propanol pathway comprises 2A, 2B, 2C, 2F and 2G. In someembodiments, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1Dand 1E; and (2) the n-propanol pathway comprises 2A, 2B, 2C, 2F and 2G.In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1K,1C, 1D and 1F; and (2) the n-propanol pathway comprises 2A, 2B, 2C, 2Fand 2G. In certain embodiments, (1) the methanol metabolic pathwaycomprises 1I; and (2) the n-propanol pathway comprises 2A, 2B, 2C, 2Fand 2G. In certain embodiments, (1) the methanol metabolic pathwaycomprises 1A, 1B, 1C, 1D, 1E and 1I; and (2) the n-propanol pathwaycomprises 2A, 2B, 2C, 2F and 2G. In some embodiments, (1) the methanolmetabolic pathway comprises 1A, 1B, 1C, 1D, 1F and 1I; and (2) then-propanol pathway comprises 2A, 2B, 2C, 2F and 2G. In some embodiments,(1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1E and 1I; and(2) the n-propanol pathway comprises 2A, 2B, 2C, 2F and 2G. In oneembodiment, (1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1Fand 1I; and (2) the n-propanol pathway comprises 2A, 2B, 2C, 2F and 2G.In another embodiment, (1) the methanol metabolic pathway comprises 1J,1L and 1I; and (2) the n-propanol pathway comprises 2A, 2B, 2C, 2F and2G. In yet another embodiment, (1) the methanol metabolic pathwaycomprises 1J, 1M, 1N, 1O and 1I; and (2) the n-propanol pathwaycomprises 2A, 2B, 2C, 2F and 2G. In certain embodiments, (1) themethanol metabolic pathway comprises 1J, 1N, 1O and 1I; and (2) then-propanol pathway comprises 2A, 2B, 2C, 2F and 2G. In some embodiments,(1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1E and 1I;and (2) the n-propanol pathway comprises 2A, 2B, 2C, 2F and 2G. In oneembodiment, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D,1F and 1I; and (2) the n-propanol pathway comprises 2A, 2B, 2C, 2F and2G. In certain embodiments, (1) the methanol metabolic pathway comprises1G; and (2) the n-propanol pathway comprises 2A, 2B, 2C, 2F and 2G. Incertain embodiments, (1) the methanol metabolic pathway comprises 1A,1B, 1C, 1D, 1E and 1G; and (2) the n-propanol pathway comprises 2A, 2B,2C, 2F and 2G. In some embodiments, (1) the methanol metabolic pathwaycomprises 1A, 1B, 1C, 1D, 1F and 1G; and (2) the n-propanol pathwaycomprises 2A, 2B, 2C, 2F and 2G. In some embodiments, (1) the methanolmetabolic pathway comprises 1J, 1C, 1D, 1E and 1G; and (2) then-propanol pathway comprises 2A, 2B, 2C, 2F and 2G. In one embodiment,(1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1F and 1G; and(2) the n-propanol pathway comprises 2A, 2B, 2C, 2F and 2G. In anotherembodiment, (1) the methanol metabolic pathway comprises 1J, 1L and 1G;and (2) the n-propanol pathway comprises 2A, 2B, 2C, 2F and 2G. In yetanother embodiment, (1) the methanol metabolic pathway comprises 1J, 1M,1N, 1O and 1G; and (2) the n-propanol pathway comprises 2A, 2B, 2C, 2Fand 2G. In certain embodiments, (1) the methanol metabolic pathwaycomprises 1J, 1N, 1O and 1G; and (2) the n-propanol pathway comprises2A, 2B, 2C, 2F and 2G. In some embodiments, (1) the methanol metabolicpathway comprises 1J, 1K, 1C, 1D, 1E and 1G; and (2) the n-propanolpathway comprises 2A, 2B, 2C, 2F and 2G. In one embodiment, (1) themethanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1F and 1G; and (2)the n-propanol pathway comprises 2A, 2B, 2C, 2F and 2G. In certainembodiments, (1) the methanol metabolic pathway comprises 1G and 1H; and(2) the n-propanol pathway comprises 2A, 2B, 2C, 2F and 2G. In certainembodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C,1D, 1E, 1G and 1H; and (2) the n-propanol pathway comprises 2A, 2B, 2C,2F and 2G. In some embodiments, (1) the methanol metabolic pathwaycomprises 1A, 1B, 1C, 1D, 1F, 1G and 1H; and (2) the n-propanol pathwaycomprises 2A, 2B, 2C, 2F and 2G. In some embodiments, (1) the methanolmetabolic pathway comprises 1J, 1C, 1D, 1E, 1G and 1H; and (2) then-propanol pathway comprises 2A, 2B, 2C, 2F and 2G. In one embodiment,(1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1F, 1G and 1H;and (2) the n-propanol pathway comprises 2A, 2B, 2C, 2F and 2G. Inanother embodiment, (1) the methanol metabolic pathway comprises 1J, 1L,1G and 1H; and (2) the n-propanol pathway comprises 2A, 2B, 2C, 2F and2G. In yet another embodiment, (1) the methanol metabolic pathwaycomprises 1J, 1M, 1N, 1O, 1G and 1H; and (2) the n-propanol pathwaycomprises 2A, 2B, 2C, 2F and 2G. In certain embodiments, (1) themethanol metabolic pathway comprises 1J, 1N, 1O, 1G and 1H; and (2) then-propanol pathway comprises 2A, 2B, 2C, 2F and 2G. In some embodiments,(1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1E, 1G and1H; and (2) the n-propanol pathway comprises 2A, 2B, 2C, 2F and 2G. Inone embodiment, (1) the methanol metabolic pathway comprises 1J, 1K, 1C,1D, 1F, 1G and 1H; and (2) the n-propanol pathway comprises 2A, 2B, 2C,2F and 2G. In some embodiments, 1K is spontaneous. In other embodiments,1K is a formaldehyde activating enzyme. In some embodiments, 1M isspontaneous. In other embodiments, 1M is a S-(hydroxymethyl)glutathionesynthase.

In one embodiment, (1) the methanol metabolic pathway comprises 1A and1B; and (2) the n-propanol pathway comprises 2A, 2D, 2E, 2F and 2G. Inanother embodiment, (1) the methanol metabolic pathway comprises 1J; and(2) the n-propanol pathway comprises 2A, 2D, 2E, 2F and 2G. In oneembodiment, (1) the methanol metabolic pathway comprises 1J and 1K; and(2) the n-propanol pathway comprises 2A, 2D, 2E, 2F and 2G. In certainembodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C,1D, and 1E; and (2) the n-propanol pathway comprises 2A, 2D, 2E, 2F and2G. In some embodiments, (1) the methanol metabolic pathway comprises1A, 1B, 1C, 1D and 1F; and (2) the n-propanol pathway comprises 2A, 2D,2E, 2F and 2G. In some embodiments, (1) the methanol metabolic pathwaycomprises 1J, 1C, 1D and 1E; and (2) the n-propanol pathway comprises2A, 2D, 2E, 2F and 2G. In one embodiment, (1) the methanol metabolicpathway comprises 1J, 1C, 1D and 1F; and (2) the n-propanol pathwaycomprises 2A, 2D, 2E, 2F and 2G. In another embodiment, (1) the methanolmetabolic pathway comprises 1J and 1L; and (2) the n-propanol pathwaycomprises 2A, 2D, 2E; 2F and 2G. In yet another embodiment, (1) themethanol metabolic pathway comprises 1J, 1M, 1N and 1O; and (2) then-propanol pathway comprises 2A, 2D, 2E, 2F and 2G. In certainembodiments, (1) the methanol metabolic pathway comprises 1J, 1N and 1O;and (2) the n-propanol pathway comprises 2A, 2D, 2E, 2F and 2G. In someembodiments, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1Dand 1E; and (2) the n-propanol pathway comprises 2A, 2D, 2E, 2F and 2G.In one embodiment, (1) the methanol metabolic pathway comprises 1J, 1K,1C, 1D and 1F; and (2) the n-propanol pathway comprises 2A, 2D, 2E, 2Fand 2G. In certain embodiments, (1) the methanol metabolic pathwaycomprises 1I; and (2) the n-propanol pathway comprises 2A, 2D, 2E, 2Fand 2G. In certain embodiments, (1) the methanol metabolic pathwaycomprises 1A, 1B, 1C, 1D, 1E and 1I; and (2) the n-propanol pathwaycomprises 2A, 2D, 2E, 2F and 2G. In some embodiments, (1) the methanolmetabolic pathway comprises 1A, 1B, 1C, 1D, 1F and 1I; and (2) then-propanol pathway comprises 2A, 2D, 2E, 2F and 2G. In some embodiments,(1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1E and 1I; and(2) the n-propanol pathway comprises 2A, 2D, 2E, 2F and 2G. In oneembodiment, (1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1Fand 1I; and (2) the n-propanol pathway comprises 2A, 2D, 2E, 2F and 2G.In another embodiment, (1) the methanol metabolic pathway comprises 1J,1L and 1I; and (2) the n-propanol pathway comprises 2A, 2D, 2E, 2F and2G. In yet another embodiment, (1) the methanol metabolic pathwaycomprises 1J, 1M, 1N, 1O and 1I; and (2) the n-propanol pathwaycomprises 2A, 2D, 2E, 2F and 2G. In certain embodiments, (1) themethanol metabolic pathway comprises 1J, 1N, 1O and 1I; and (2) then-propanol pathway comprises 2A, 2D, 2E, 2F and 2G. In some embodiments,(1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1E and 1I;and (2) the n-propanol pathway comprises 2A, 2D, 2E, 2F and 2G. In oneembodiment, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D,1F and 1I; and (2) the n-propanol pathway comprises 2A, 2D, 2E, 2F and2G. In certain embodiments, (1) the methanol metabolic pathway comprises1G; and (2) the n-propanol pathway comprises 2A, 2D, 2E, 2F and 2G. Incertain embodiments, (1) the methanol metabolic pathway comprises 1A,1B, 1C, 1D, 1E and 1G; and (2) the n-propanol pathway comprises 2A, 2D,2E, 2F and 2G. In some embodiments, (1) the methanol metabolic pathwaycomprises 1A, 1B, 1C, 1D, 1F and 1G; and (2) the n-propanol pathwaycomprises 2A, 2D, 2E, 2F and 2G. In some embodiments, (1) the methanolmetabolic pathway comprises 1J, 1C, 1D, 1E and 1G; and (2) then-propanol pathway comprises 2A, 2D, 2E, 2F and 2G. In one embodiment,(1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1F and 1G; and(2) the n-propanol pathway comprises 2A, 2D, 2E, 2F and 2G. In anotherembodiment, (1) the methanol metabolic pathway comprises 1J, 1L and 1G;and (2) the n-propanol pathway comprises 2A, 2D, 2E, 2F and 2G. In yetanother embodiment, (1) the methanol metabolic pathway comprises 1J, 1M,1N, 1O and 1G; and (2) the n-propanol pathway comprises 2A, 2D, 2E, 2Fand 2G. In certain embodiments, (1) the methanol metabolic pathwaycomprises 1J, 1N, 1O and 1G; and (2) the n-propanol pathway comprises2A, 2D, 2E, 2F and 2G. In some embodiments, (1) the methanol metabolicpathway comprises 1J, 1K, 1C, 1D, 1E and 1G; and (2) the n-propanolpathway comprises 2A, 2D, 2E, 2F and 2G. In one embodiment, (1) themethanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1F and 1G; and (2)the n-propanol pathway comprises 2A, 2D, 2E, 2F and 2G. In certainembodiments, (1) the methanol metabolic pathway comprises 1G and 1H; and(2) the n-propanol pathway comprises 2A, 2D, 2E, 2F and 2G. In certainembodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C,1D, 1E, 1G and 1H; and (2) the n-propanol pathway comprises 2A, 2D, 2E,2F and 2G. In some embodiments, (1) the methanol metabolic pathwaycomprises 1A, 1B, 1C, 1D, 1F, 1G and 1H; and (2) the n-propanol pathwaycomprises 2A, 2D, 2E, 2F and 2G. In some embodiments, (1) the methanolmetabolic pathway comprises 1J, 1C, 1D, 1E, 1G and 1H; and (2) then-propanol pathway comprises 2A, 2D, 2E, 2F and 2G. In one embodiment,(1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1F, 1G and 1H;and (2) the n-propanol pathway comprises 2A, 2D, 2E, 2F and 2G. Inanother embodiment, (1) the methanol metabolic pathway comprises 1J, 1L,1G and 1H; and (2) the n-propanol pathway comprises 2A, 2D, 2E, 2F and2G. In yet another embodiment, (1) the methanol metabolic pathwaycomprises 1J, 1M, 1N, 1O, 1G and 1H; and (2) the n-propanol pathwaycomprises 2A, 2D, 2E, 2F and 2G. In certain embodiments, (1) themethanol metabolic pathway comprises 1J, 1N, 1O, 1G and 1H; and (2) then-propanol pathway comprises 2A, 2D, 2E, 2F and 2G. In some embodiments,(1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1E, 1G and1H; and (2) the n-propanol pathway comprises 2A, 2D, 2E, 2F and 2G. Inone embodiment, (1) the methanol metabolic pathway comprises 1J, 1K, 1C,1D, 1F, 1G and 1H; and (2) the n-propanol pathway comprises 2A, 2D, 2E,2F and 2G. In some embodiments, 1K is spontaneous. In other embodiments,1K is a formaldehyde activating enzyme. In some embodiments, 1M isspontaneous. In other embodiments, 1M is a S-(hydroxymethyl)glutathionesynthase.

In one embodiment, the non-naturally occurring microbial organismcomprises (1) a methanol metabolic pathway comprising 1A and 1B; 1J; 1Jand 1K; 1A, 1B, 1C, 1D, and 1E; 1A, 1B, 1C, 1D and 1F; 1J, 1C, 1D and1E; 1J, 1C, 1D and 1F; 1J and 1L; 1J, 1M, 1N and 1O; 1J, 1N and 1O; 1J,1K, 1C, 1D and 1E; 1J, 1K, 1C, 1D and 1F; 1I; 1A, 1B, 1C, 1D, 1E and 1I;1A, 1B, 1C, 1D, 1F and 1I; 1J, 1C, 1D, 1E and 1I; 1J, 1C, 1D, 1F and 1I;1J, 1L and 1I; 1J, 1M, 1N, 1O and 1I; 1J, 1N, 1O and 1I; 1J, 1K, 1C, 1D,1E and 1I; 1J, 1K, 1C, 1D, 1F and 1I; 1G; 1A, 1B, 1C, 1D, 1E and 1G; 1A,1B, 1C, 1D, 1F and 1G; 1J, 1C, 1D, 1E and 1G; 1J, 1C, 1D, 1F and 1G; 1J,1L and 1G; 1J, 1M, 1N, 1O and 1G; 1J, 1N, 1O and 1G; 1J, 1K, 1C, 1D, 1Eand 1G; 1J, 1K, 1C, 1D, 1F and 1G; 1G and 1H; 1A, 1B, 1C, 1D, 1E, 1G and1H; 1A, 1B, 1C, 1D, 1F, 1G and 1H; 1J, 1C, 1D, 1E, 1G and 1H; 1J, 1C,1D, 1F, 1G and 1H; 1J, 1L, 1G and 1H; 1J, 1M, 1N, 1O, 1G and 1H; 1J, 1N,1O, 1G and 1H; 1J, 1K, 1C, 1D, 1E, 1G and 1H; or 1J, 1K, 1C, 1D, 1F, 1Gand 1H; and (2) a n-propanol pathway. In some embodiments, 1K isspontaneous. In other embodiments, 1K is a formaldehyde activatingenzyme. In some embodiments, 1M is spontaneous. In other embodiments, 1Mis a S-(hydroxymethyl)glutathione synthase.

Any methanol metabolic pathway provided herein can be combined with anyn-propanol pathway provided herein.

Exemplary enzymes for the conversion of glucose to 1,3-propanediol(e.g., via glyceraldehydes-3-phosphate or dihydroxyacetone phosphate)include a glyceraldehyde-3-phosphate reductase (FIG. 3, step A); aglycerol-3-phosphate phosphatase or a glycerol kinase (FIG. 3, step B);a glycerol dehydratase (FIG. 3, step C); a 3-hydroxypropanal reductase(FIG. 3, step D); a dihydroxyacetone phosphate phosphatase or adihydroxyacetone kinase (FIG. 3, step E); a dihydroxyacetone reductase(FIG. 3, step F); and a dihydroxyacetone phosphate reductase (FIG. 3,step G).

In another aspect, provided herein is a non-naturally occurringmicrobial organism, comprising (1) a methanol metabolic pathway, whereinsaid organism comprises at least one exogenous nucleic acid encoding amethanol metabolic pathway enzyme in a sufficient amount to enhance theavailability of reducing equivalents in the presence of methanol; and(2) an 1,3-propanediol pathway, wherein said organism comprises at leastone exogenous nucleic acid encoding an 1,3-propanediol pathway enzymeexpressed in a sufficient amount to produce 1,3-propanediol. In oneembodiment, the at least one exogenous nucleic acid encoding themethanol metabolic pathway enzyme enhances the availability of reducingequivalents in the presence of methanol in a sufficient amount toincrease the amount of 1,3-propanediol produced by the non-naturallymicrobial organism. In some embodiments, the methanol metabolic pathwaycomprises any of the various combinations of methanol metabolic pathwayenzymes described above or elsewhere herein.

In certain embodiments, (1) the methanol metabolic pathway comprises:1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 1I, 1J, 1K, 1L, 1M, 1N, or 1O or anycombination of 1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 1I, 1J, 1K, 1L, 1M, 1N,or 1O, thereof, wherein 1A is a methanol methyltransferase; 1B is amethylenetetrahydrofolate reductase; 1C is a methylenetetrahydrofolatedehydrogenase; 1D is a methenyltetrahydrofolate cyclohydrolase; 1E is aformyltetrahydrofolate deformylase; 1F is a formyltetrahydrofolatesynthetase; 1G is a formate hydrogen lyase; 1H is a hydrogenase, 1I is aformate dehydrogenase; 1J is a methanol dehydrogenase; 1K is spontaneousor formaldehyde activating enzyme; 1L is a formaldehyde dehydrogenase;1M is spontaneous or a S-(hydroxymethyl)glutathione synthase; 1N isglutathione-dependent formaldehyde dehydrogenase and 1O isS-formylglutathione hydrolase; and (2) the 1,3-propanediol pathwaycomprises 3A, 3B, 3C, 3D, 3E, 3F or 3G, or any combination thereof,wherein 3A is a glyceraldehyde-3-phosphate reductase; 3B is aglycerol-3-phosphate phosphatase or a glycerol kinase; 3C is a glyceroldehydratase; 3D is a 3-hydroxypropanal reductase; 3E is adihydroxyacetone phosphate phosphatase or a dihydroxyacetone kinase; 3Fis a dihydroxyacetone reductase; and 3G is a dihydroxyacetone phosphatereductase. In some embodiments, 1K is spontaneous. In other embodiments,1K is a formaldehyde activating enzyme. In some embodiments, 1M isspontaneous. In other embodiments, 1M is a S-(hydroxymethyl)glutathionesynthase. In some embodiments, 3B is a glycerol-3-phosphate phosphatase.In other embodiments, 3C is a glycerol kinase. In some embodiments, 3Eis a dihydroxyacetone phosphate phosphatase. In other embodiments, 3E isa dihydroxyacetone kinase.

In one embodiment, the 1,3-propanediol pathway comprises 3A. In anotherembodiment, the 1,3-propanediol pathway comprises 3B. In an embodiment,the 1,3-propanediol pathway comprises 3C. In another embodiment, the1,3-propanediol pathway comprises 3D. In one embodiment, the1,3-propanediol pathway comprises 3E. In yet another embodiment, the1,3-propanediol pathway comprises 3F. In another embodiment, the1,3-propanediol pathway comprises 3G. Any combination of two, three,four, five, six or seven 1,3-propanediol pathway enzymes 3A, 3B, 3C, 3D,3E, 3F and 3G is also contemplated.

In some embodiments, the methanol metabolic pathway is a methanolmetabolic pathway depicted in FIG. 1, and the 1,3-propanediol pathway isan 1,3-propanediol pathway depicted in FIG. 3.

Exemplary sets of 1,3-propanediol pathway enzymes to convert glucose to1,3-propanediol (e.g., via glyceraldehydes-3-phosphate ordihydroxyacetone phosphate) according to FIG. 3, include (i) 3A, 3B, 3Cand 3D; (ii) 3G, 3B, 3C and 3D; (iii) 3E, 3F, 3C and 3D.

In one embodiment, (1) the methanol metabolic pathway comprises 1A and1B; and (2) the 1,3-propanediol pathway comprises 3A, 3B, 3C and 3D. Inanother embodiment, (1) the methanol metabolic pathway comprises 1J; and(2) the 1,3-propanediol pathway comprises 3A, 3B, 3C and 3D. In oneembodiment, (1) the methanol metabolic pathway comprises 1J and 1K; and(2) the 1,3-propanediol pathway comprises 3A, 3B, 3C and 3D. In certainembodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C,1D, and 1E; and (2) the 1,3-propanediol pathway comprises 3A, 3B, 3C and3D. In some embodiments, (1) the methanol metabolic pathway comprises1A, 1B, 1C, 1D and 1F; and (2) the 1,3-propanediol pathway comprises 3A,3B, 3C and 3D. In some embodiments, (1) the methanol metabolic pathwaycomprises 1J, 1C, 1D and 1E; and (2) the 1,3-propanediol pathwaycomprises 3A, 3B, 3C and 3D. In one embodiment, (1) the methanolmetabolic pathway comprises 1J, 1C, 1D and 1F; and (2) the1,3-propanediol pathway comprises 3A, 3B, 3C and 3D. In anotherembodiment, (1) the methanol metabolic pathway comprises 1J and 1L; and(2) the 1,3-propanediol pathway comprises 3A, 3B, 3C and 3D. In yetanother embodiment, (1) the methanol metabolic pathway comprises 1J, 1M,1N and 1O; and (2) the 1,3-propanediol pathway comprises 3A, 3B, 3C and3D. In certain embodiments, (1) the methanol metabolic pathway comprises1J, 1N and 1O; and (2) the 1,3-propanediol pathway comprises 3A, 3B, 3Cand 3D. In some embodiments, (1) the methanol metabolic pathwaycomprises 1J, 1K, 1C, 1D and 1E; and (2) the 1,3-propanediol pathwaycomprises 3A, 3B, 3C and 3D. In one embodiment, (1) the methanolmetabolic pathway comprises 1J, 1K, 1C, 1D and 1E; and (2) the1,3-propanediol pathway comprises 3A, 3B, 3C and 3D. In certainembodiments, (1) the methanol metabolic pathway comprises 1I; and (2)the 1,3-propanediol pathway comprises 3A, 3B, 3C and 3D. In certainembodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C,1D, 1E and 1I; and (2) the 1,3-propanediol pathway comprises 3A, 3B, 3Cand 3D. In some embodiments, (1) the methanol metabolic pathwaycomprises 1A, 1B, 1C, 1D, 1F and 1I; and (2) the 1,3-propanediol pathwaycomprises 3A, 3B, 3C and 3D. In some embodiments, (1) the methanolmetabolic pathway comprises 1J, 1C, 1D, 1E and 1I; and (2) the1,3-propanediol pathway comprises 3A, 3B, 3C and 3D. In one embodiment,(1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1F and 1I; and(2) the 1,3-propanediol pathway comprises 3A, 3B, 3C and 3D. In anotherembodiment, (1) the methanol metabolic pathway comprises 1J, 1L and 1I;and (2) the 1,3-propanediol pathway comprises 3A, 3B, 3C and 3D. In yetanother embodiment, (1) the methanol metabolic pathway comprises 1J, 1M,1N, 1O and 1I; and (2) the 1,3-propanediol pathway comprises 3A, 3B, 3Cand 3D. In certain embodiments, (1) the methanol metabolic pathwaycomprises 1J, 1N, 1O and 1I; and (2) the 1,3-propanediol pathwaycomprises 3A, 3B, 3C and 3D. In some embodiments, (1) the methanolmetabolic pathway comprises 1J, 1K, 1C, 1D, 1E and 1I; and (2) the1,3-propanediol pathway comprises 3A, 3B, 3C and 3D. In one embodiment,(1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1F and 1I;and (2) the 1,3-propanediol pathway comprises 3A, 3B, 3C and 3D. Incertain embodiments, (1) the methanol metabolic pathway comprises 1G;and (2) the 1,3-propanediol pathway comprises 3A, 3B, 3C and 3D. Incertain embodiments, (1) the methanol metabolic pathway comprises 1A,1B, 1C, 1D, 1E and 1G; and (2) the 1,3-propanediol pathway comprises 3A,3B, 3C and 3D. In some embodiments, (1) the methanol metabolic pathwaycomprises 1A, 1B, 1C, 1D, 1F and 1G; and (2) the 1,3-propanediol pathwaycomprises 3A, 3B, 3C and 3D. In some embodiments, (1) the methanolmetabolic pathway comprises 1J, 1C, 1D, 1E and 1G; and (2) the1,3-propanediol pathway comprises 3A, 3B, 3C and 3D. In one embodiment,(1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1F and 1G; and(2) the 1,3-propanediol pathway comprises 3A, 3B, 3C and 3D. In anotherembodiment, (1) the methanol metabolic pathway comprises 1J, 1L and 1G;and (2) the 1,3-propanediol pathway comprises 3A, 3B, 3C and 3D. In yetanother embodiment, (1) the methanol metabolic pathway comprises 1J, 1M,1N, 1O and 1G; and (2) the 1,3-propanediol pathway comprises 3A, 3B, 3Cand 3D. In certain embodiments, (1) the methanol metabolic pathwaycomprises 1J, 1N, 1O and 1G; and (2) the 1,3-propanediol pathwaycomprises 3A, 3B, 3C and 3D. In some embodiments, (1) the methanolmetabolic pathway comprises 1J, 1K, 1C, 1D, 1E and 1G; and (2) the1,3-propanediol pathway comprises 3A, 3B, 3C and 3D. In one embodiment,(1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1F and 1G;and (2) the 1,3-propanediol pathway comprises 3A, 3B, 3C and 3D. Incertain embodiments, (1) the methanol metabolic pathway comprises 1G and1H; and (2) the 1,3-propanediol pathway comprises 3A, 3B, 3C and 3D. Incertain embodiments, (1) the methanol metabolic pathway comprises 1A,1B, 1C, 1D, 1E, 1G and 1H; and (2) the 1,3-propanediol pathway comprises3A, 3B, 3C and 3D. In some embodiments, (1) the methanol metabolicpathway comprises 1A, 1B, 1C, 1D, 1F, 1G and 1H; and (2) the1,3-propanediol pathway comprises 3A, 3B, 3C and 3D. In someembodiments, (1) the methanol metabolic pathway comprises 1J, 1C, 1D,1E, 1G and 1H; and (2) the 1,3-propanediol pathway comprises 3A, 3B, 3Cand 3D. In one embodiment, (1) the methanol metabolic pathway comprises1J, 1C, 1D, 1F, 1G and 1H; and (2) the 1,3-propanediol pathway comprises3A, 3B, 3C and 3D. In another embodiment, (1) the methanol metabolicpathway comprises 1J, 1L, 1G and 1H; and (2) the 1,3-propanediol pathwaycomprises 3A, 3B, 3C and 3D. In yet another embodiment, (1) the methanolmetabolic pathway comprises 1J, 1M, 1N, 1O, 1G and 1H; and (2) the1,3-propanediol pathway comprises 3A, 3B, 3C and 3D. In certainembodiments, (1) the methanol metabolic pathway comprises 1J, 1N, 1O, 1Gand 1H; and (2) the 1,3-propanediol pathway comprises 3A, 3B, 3C and 3D.In some embodiments, (1) the methanol metabolic pathway comprises 1J,1K, 1C, 1D, 1E, 1G and 1H; and (2) the 1,3-propanediol pathway comprises3A, 3B, 3C and 3D. In one embodiment, (1) the methanol metabolic pathwaycomprises 1J, 1K, 1C, 1D, 1F, 1G and 1H; and (2) the 1,3-propanediolpathway comprises 3A, 3B, 3C and 3D. In some embodiments, 1K isspontaneous. In other embodiments, 1K is a formaldehyde activatingenzyme. In some embodiments, 1M is spontaneous. In other embodiments, 1Mis a S-(hydroxymethyl)glutathione synthase. In some embodiments, 3B is aglycerol-3-phosphate phosphatase. In other embodiments, 3C is a glycerolkinase. In some embodiments, 3E is a dihydroxyacetone phosphatephosphatase. In other embodiments, 3E is a dihydroxyacetone kinase.

In one embodiment, (1) the methanol metabolic pathway comprises 1A and1B; and (2) the 1,3-propanediol pathway comprises 3G, 3B, 3C and 3D. Inanother embodiment, (1) the methanol metabolic pathway comprises 1J; and(2) the 1,3-propanediol pathway comprises 3G, 3B, 3C and 3D. In oneembodiment, (1) the methanol metabolic pathway comprises 1J and 1K; and(2) the 1,3-propanediol pathway comprises 3G, 3B, 3C and 3D. In certainembodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C,1D, and 1E; and (2) the 1,3-propanediol pathway comprises 3G, 3B, 3C and3D. In some embodiments, (1) the methanol metabolic pathway comprises1A, 1B, 1C, 1D and 1F; and (2) the 1,3-propanediol pathway comprises 3G,3B, 3C and 3D. In some embodiments, (1) the methanol metabolic pathwaycomprises 1J, 1C, 1D and 1E; and (2) the 1,3-propanediol pathwaycomprises 3G, 3B, 3C and 3D. In one embodiment, (1) the methanolmetabolic pathway comprises 1J, 1C, 1D and 1F; and (2) the1,3-propanediol pathway comprises 3G, 3B, 3C and 3D. In anotherembodiment, (1) the methanol metabolic pathway comprises 1J and 1L; and(2) the 1,3-propanediol pathway comprises 3G, 3B, 3C and 3D. In yetanother embodiment, (1) the methanol metabolic pathway comprises 1J, 1M,1N and 1O; and (2) the 1,3-propanediol pathway comprises 3G, 3B, 3C and3D. In certain embodiments, (1) the methanol metabolic pathway comprises1J, 1N and 1O; and (2) the 1,3-propanediol pathway comprises 3G, 3B, 3Cand 3D. In some embodiments, (1) the methanol metabolic pathwaycomprises 1J, 1K, 1C, 1D and 1E; and (2) the 1,3-propanediol pathwaycomprises 3G, 3B, 3C and 3D. In one embodiment, (1) the methanolmetabolic pathway comprises 1J, 1K, 1C, 1D and 1F; and (2) the1,3-propanediol pathway comprises 3G, 3B, 3C and 3D. In certainembodiments, (1) the methanol metabolic pathway comprises 1I; and (2)the 1,3-propanediol pathway comprises 3G, 3B, 3C and 3D. In certainembodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C,1D, 1E and 1I; and (2) the 1,3-propanediol pathway comprises 3G, 3B, 3Cand 3D. In some embodiments, (1) the methanol metabolic pathwaycomprises 1A, 1B, 1C, 1D, 1F and 1I; and (2) the 1,3-propanediol pathwaycomprises 3G, 3B, 3C and 3D. In some embodiments, (1) the methanolmetabolic pathway comprises 1J, 1C, 1D, 1E and 1I; and (2) the1,3-propanediol pathway comprises 3G, 3B, 3C and 3D. In one embodiment,(1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1F and 1I; and(2) the 1,3-propanediol pathway comprises 3G, 3B, 3C and 3D. In anotherembodiment, (1) the methanol metabolic pathway comprises 1J, 1L and 1I;and (2) the 1,3-propanediol pathway comprises 3G, 3B, 3C and 3D. In yetanother embodiment, (1) the methanol metabolic pathway comprises 1J, 1M,1N, 1O and 1I; and (2) the 1,3-propanediol pathway comprises 3G, 3B, 3Cand 3D. In certain embodiments, (1) the methanol metabolic pathwaycomprises 1J, 1N, 1O and 1I; and (2) the 1,3-propanediol pathwaycomprises 3G, 3B, 3C and 3D. In some embodiments, (1) the methanolmetabolic pathway comprises 1J, 1K, 1C, 1D, 1E and 1I; and (2) the1,3-propanediol pathway comprises 3G, 3B, 3C and 3D. In one embodiment,(1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1F and 1I;and (2) the 1,3-propanediol pathway comprises 3G, 3B, 3C and 3D. Incertain embodiments, (1) the methanol metabolic pathway comprises 1G;and (2) the 1,3-propanediol pathway comprises 3G, 3B, 3C and 3D. Incertain embodiments, (1) the methanol metabolic pathway comprises 1A,1B, 1C, 1D, 1E and 1G; and (2) the 1,3-propanediol pathway comprises 3G,3B, 3C and 3D. In some embodiments, (1) the methanol metabolic pathwaycomprises 1A, 1B, 1C, 1D, 1F and 1G; and (2) the 1,3-propanediol pathwaycomprises 3G, 3B, 3C and 3D. In some embodiments, (1) the methanolmetabolic pathway comprises 1J, 1C, 1D, 1E and 1G; and (2) the1,3-propanediol pathway comprises 3G, 3B, 3C and 3D. In one embodiment,(1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1F and 1G; and(2) the 1,3-propanediol pathway comprises 3G, 3B, 3C and 3D. In anotherembodiment, (1) the methanol metabolic pathway comprises 1J, 1L and 1G;and (2) the 1,3-propanediol pathway comprises 3G, 3B, 3C and 3D. In yetanother embodiment, (1) the methanol metabolic pathway comprises 1J, 1M,1N, 1O and 1G; and (2) the 1,3-propanediol pathway comprises 3G, 3B, 3Cand 3D. In certain embodiments, (1) the methanol metabolic pathwaycomprises 1J, 1N, 1O and 1G; and (2) the 1,3-propanediol pathwaycomprises 3G, 3B, 3C and 3D. In some embodiments, (1) the methanolmetabolic pathway comprises 1J, 1K, 1C, 1D, 1E and 1G; and (2) the1,3-propanediol pathway comprises 3G, 3B, 3C and 3D. In one embodiment,(1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1F and 1G;and (2) the 1,3-propanediol pathway comprises 3G, 3B, 3C and 3D. Incertain embodiments, (1) the methanol metabolic pathway comprises 1G and1H; and (2) the 1,3-propanediol pathway comprises 3G, 3B, 3C and 3D. Incertain embodiments, (1) the methanol metabolic pathway comprises 1A,1B, 1C, 1D, 1E, 1G and 1H; and (2) the 1,3-propanediol pathway comprises3G, 3B, 3C and 3D. In some embodiments, (1) the methanol metabolicpathway comprises 1A, 1B, 1C, 1D, 1F, 1G and 1H; and (2) the1,3-propanediol pathway comprises 3G, 3B, 3C and 3D. In someembodiments, (1) the methanol metabolic pathway comprises 1J, 1C, 1D,1E, 1G and 1H; and (2) the 1,3-propanediol pathway comprises 3G, 3B, 3Cand 3D. In one embodiment, (1) the methanol metabolic pathway comprises1J, 1C, 1D, 1F, 1G and 1H; and (2) the 1,3-propanediol pathway comprises3G, 3B, 3C and 3D. In another embodiment, (1) the methanol metabolicpathway comprises 1J, 1I, 1G and 1H; and (2) the 1,3-propanediol pathwaycomprises 3G, 3B, 3C and 3D. In yet another embodiment, (1) the methanolmetabolic pathway comprises 1J, 1M, 1N, 1O, 1G and 1H; and (2) the1,3-propanediol pathway comprises 3G, 3B, 3C and 3D. In certainembodiments, (1) the methanol metabolic pathway comprises 1J, 1N, 1O, 1Gand 1H; and (2) the 1,3-propanediol pathway comprises 3G, 3B, 3C and 3D.In some embodiments, (1) the methanol metabolic pathway comprises 1J,1K, 1C, 1D, 1E, 1G and 1H; and (2) the 1,3-propanediol pathway comprises3G, 3B, 3C and 3D. In one embodiment, (1) the methanol metabolic pathwaycomprises 1J, 1K, 1C, 1D, 1F, 1G and 1H; and (2) the 1,3-propanediolpathway comprises 3G, 3B, 3C and 3D. In some embodiments, 1K isspontaneous. In other embodiments, 1K is a formaldehyde activatingenzyme. In some embodiments, 1M is spontaneous. In other embodiments, 1Mis a S-(hydroxymethyl)glutathione synthase. In some embodiments, 3B is aglycerol-3-phosphate phosphatase. In other embodiments, 3C is a glycerolkinase. In some embodiments, 3E is a dihydroxyacetone phosphatephosphatase. In other embodiments, 3E is a dihydroxyacetone kinase.

In one embodiment, (1) the methanol metabolic pathway comprises 1A and1B; and (2) the 1,3-propanediol pathway comprises 3E, 3F, 3C and 3D. Inanother embodiment, (1) the methanol metabolic pathway comprises 1J; and(2) the 1,3-propanediol pathway comprises 3E, 3F, 3C and 3D. In oneembodiment, (1) the methanol metabolic pathway comprises 1J and 1K; and(2) the 1,3-propanediol pathway comprises 3E, 3F, 3C and 3D. In certainembodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C,1D, and 1E; and (2) the 1,3-propanediol pathway comprises 3E, 3F, 3C and3D. In some embodiments, (1) the methanol metabolic pathway comprises1A, 1B, 1C, 1D and 1F; and (2) the 1,3-propanediol pathway comprises 3E,3F, 3C and 3D. In some embodiments, (1) the methanol metabolic pathwaycomprises 1J, 1C, 1D and 1E; and (2) the 1,3-propanediol pathwaycomprises 3E, 3F, 3C and 3D. In one embodiment, (1) the methanolmetabolic pathway comprises 1J, 1C, 1D and 1F; and (2) the1,3-propanediol pathway comprises 3E, 3F, 3C and 3D. In anotherembodiment, (1) the methanol metabolic pathway comprises 1J and 1L; and(2) the 1,3-propanediol pathway comprises 3E, 3F, 3C and 3D. In yetanother embodiment, (1) the methanol metabolic pathway comprises 1J, 1M,1N and 1O; and (2) the 1,3-propanediol pathway comprises 3E, 3F, 3C and3D. In certain embodiments, (1) the methanol metabolic pathway comprises1J, 1N and 1O; and (2) the 1,3-propanediol pathway comprises 3E, 3F, 3Cand 3D. In some embodiments, (1) the methanol metabolic pathwaycomprises 1J, 1K, 1C, 1D and 1E; and (2) the 1,3-propanediol pathwaycomprises 3E, 3F, 3C and 3D. In one embodiment, (1) the methanolmetabolic pathway comprises 1J, 1K, 1C, 1D and 1F; and (2) the1,3-propanediol pathway comprises 3E, 3F, 3C and 3D. In certainembodiments, (1) the methanol metabolic pathway comprises 1I; and (2)the 1,3-propanediol pathway comprises 3E, 3F, 3C and 3D. In certainembodiments, (1) the methanol metabolic pathway comprises 1A, 1B, 1C,1D, 1E and 1I; and (2) the 1,3-propanediol pathway comprises 3E, 3F, 3Cand 3D. In some embodiments, (1) the methanol metabolic pathwaycomprises 1A, 1B, 1C, 1D, 1F and 1I; and (2) the 1,3-propanediol pathwaycomprises 3E, 3F, 3C and 3D. In some embodiments, (1) the methanolmetabolic pathway comprises 1J, 1C, 1D, 1E and 1I; and (2) the1,3-propanediol pathway comprises 3E, 3F, 3C and 3D. In one embodiment,(1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1F and 1I; and(2) the 1,3-propanediol pathway comprises 3E, 3F, 3C and 3D. In anotherembodiment, (1) the methanol metabolic pathway comprises 1J, 1L and 1I;and (2) the 1,3-propanediol pathway comprises 3E, 3F, 3C and 3D. In yetanother embodiment, (1) the methanol metabolic pathway comprises 1J, 1M,1N, 1O and 1I; and (2) the 1,3-propanediol pathway comprises 3E, 3F, 3Cand 3D. In certain embodiments, (1) the methanol metabolic pathwaycomprises 1J, 1N, 1O and 1I; and (2) the 1,3-propanediol pathwaycomprises 3E, 3F, 3C and 3D. In some embodiments, (1) the methanolmetabolic pathway comprises 1J, 1K, 1C, 1D, 1E and 1I; and (2) the1,3-propanediol pathway comprises 3E, 3F, 3C and 3D. In one embodiment,(1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1F and 1I;and (2) the 1,3-propanediol pathway comprises 3E, 3F, 3C and 3D. Incertain embodiments, (1) the methanol metabolic pathway comprises 1G;and (2) the 1,3-propanediol pathway comprises 3E, 3F, 3C and 3D. Incertain embodiments, (1) the methanol metabolic pathway comprises 1A,1B, 1C, 1D, 1E and 1G; and (2) the 1,3-propanediol pathway comprises 3E,3F, 3C and 3D. In some embodiments, (1) the methanol metabolic pathwaycomprises 1A, 1B, 1C, 1D, 1F and 1G; and (2) the 1,3-propanediol pathwaycomprises 3E, 3F, 3C and 3D. In some embodiments, (1) the methanolmetabolic pathway comprises 1J, 1C, 1D, 1E and 1G; and (2) the1,3-propanediol pathway comprises 3E, 3F, 3C and 3D. In one embodiment,(1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1F and 1G; and(2) the 1,3-propanediol pathway comprises 3E, 3F, 3C and 3D. In anotherembodiment, (1) the methanol metabolic pathway comprises 1J, 1L and 1G;and (2) the 1,3-propanediol pathway comprises 3E, 3F, 3C and 3D. In yetanother embodiment, (1) the methanol metabolic pathway comprises 1J, 1M,1N, 1O and 1G; and (2) the 1,3-propanediol pathway comprises 3E, 3F, 3Cand 3D. In certain embodiments, (1) the methanol metabolic pathwaycomprises 1J, 1N, 1O and 1G; and (2) the 1,3-propanediol pathwaycomprises 3E, 3F, 3C and 3D. In some embodiments, (1) the methanolmetabolic pathway comprises 1J, 1K, 1C, 1D, 1E and 1G; and (2) the1,3-propanediol pathway comprises 3E, 3F, 3C and 3D. In one embodiment,(1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1F and 1G;and (2) the 1,3-propanediol pathway comprises 3E, 3F, 3C and 3D. Incertain embodiments, (1) the methanol metabolic pathway comprises 1G and1H; and (2) the 1,3-propanediol pathway comprises 3E, 3F, 3C and 3D. Incertain embodiments, (1) the methanol metabolic pathway comprises 1A,1B, 1C, 1D, 1E, 1G and 1H; and (2) the 1,3-propanediol pathway comprises3E, 3F, 3C and 3D. In some embodiments, (1) the methanol metabolicpathway comprises 1A, 1B, 1C, 1D, 1F, 1G and 1H; and (2) the1,3-propanediol pathway comprises 3E, 3F, 3C and 3D. In someembodiments, (1) the methanol metabolic pathway comprises 1J, 1C, 1D,1E, 1G and 1H; and (2) the 1,3-propanediol pathway comprises 3E, 3F, 3Cand 3D. In one embodiment, (1) the methanol metabolic pathway comprises1J, 1C, 1D, 1F, 1G and 1H; and (2) the 1,3-propanediol pathway comprises3E, 3F, 3C and 3D. In another embodiment, (1) the methanol metabolicpathway comprises 1J, 1L, 1G and 1H; and (2) the 1,3-propanediol pathwaycomprises 3E, 3F, 3C and 3D. In yet another embodiment, (1) the methanolmetabolic pathway comprises 1J, 1M, 1N, 1O, 1G and 1H; and (2) the1,3-propanediol pathway comprises 3E, 3F, 3C and 3D. In certainembodiments, (1) the methanol metabolic pathway comprises 1J, 1N, 1O, 1Gand 1H; and (2) the 1,3-propanediol pathway comprises 3E, 3F, 3C and 3D.In some embodiments, (1) the methanol metabolic pathway comprises 1J,1K, 1C, 1D, 1E, 1G and 1H; and (2) the 1,3-propanediol pathway comprises3E, 3F, 3C and 3D. In one embodiment, (1) the methanol metabolic pathwaycomprises 1J, 1K, 1C, 1D, 1F, 1G and 1H; and (2) the 1,3-propanediolpathway comprises 3E, 3F, 3C and 3D. In some embodiments, 1K isspontaneous. In other embodiments, 1K is a formaldehyde activatingenzyme. In some embodiments, 1M is spontaneous. In other embodiments, 1Mis a S-(hydroxymethyl)glutathione synthase. In some embodiments, 3B is aglycerol-3-phosphate phosphatase. In other embodiments, 3C is a glycerolkinase. In some embodiments, 3E is a dihydroxyacetone phosphatephosphatase. In other embodiments, 3E is a dihydroxyacetone kinase.

In one embodiment, the non-naturally occurring microbial organismcomprises (1) a methanol metabolic pathway comprising 1A and 1B; 1J; 1Jand 1K; 1A, 1B, 1C, 1D, and 1E; 1A, 1B, 1C, 1D and 1F; 1J, 1C, 1D and1E; 1J, 1C, 1D and 1F; 1J and 1L; 1J, 1M, 1N and 1O; 1J, 1N and 1O; 1J,1K, 1C, 1D and 1E; 1J, 1K, 1C, 1D and 1F; 1I; 1A, 1B, 1C, 1D, 1E and 1I;1A, 1B, 1C, 1D, 1F and 1I; 1J, 1C, 1D, 1E and 1I; 1J, 1C, 1D, 1F and 1I;1J, 1L and 1I; 1J, 1M, 1N, 1O and 1I; 1J, 1N, 1O and 1I; 1J, 1K, 1C, 1D,1E and 1I; 1J, 1K, 1C, 1D, 1F and 1I; 1G; 1A, 1B, 1C, 1D, 1E and 1G; 1A,1B, 1C, 1D, 1F and 1G; 1J, 1C, 1D, 1E and 1G; 1J, 1C, 1D, 1F and 1G; 1J,1L and 1G; 1J, 1M, 1N, 1O and 1G; 1J, 1N, 1O and 1G; 1J, 1K, 1C, 1D, 1Eand 1G; 1J, 1K, 1C, 1D, 1F and 1G; 1G and 1H; 1A, 1B, 1C, 1D, 1E, 1G and1H; 1A, 1B, 1C, 1D, 1F, 1G and 1H; 1J, 1C, 1D, 1E, 1G and 1H; 1J, 1C,1D, 1F, 1G and 1H; 1J, 1L, 1G and 1H; 1J, 1M, 1N, 1O, 1G and 1H; 1J, 1N,1O, 1G and 1H; 1J, 1K, 1C, 1D, 1E, 1G and 1H; or 1J, 1K, 1C, 1D, 1F, 1Gand 1H; and (2) a 1,3-propanediol pathway. In some embodiments, 1K isspontaneous. In other embodiments, 1K is a formaldehyde activatingenzyme. In some embodiments, 1M is spontaneous. In other embodiments, 1Mis a S-(hydroxymethyl)glutathione synthase.

Any methanol metabolic pathway provided herein can be combined with any1,3-propanediol pathway provided herein.

Exemplary enzymes for the conversion of glucose to glycerol (e.g., viaglyceraldehydes-3-phosphate or dihydroxyacetone phosphate) include aglyceraldehyde-3-phosphate reductase (FIG. 3, step A); aglycerol-3-phosphate phosphatase or a glycerol kinase (FIG. 3, step B);a dihydroxyacetone phosphate phosphatase or a dihydroxyacetone kinase(FIG. 3, step E); a dihydroxyacetone reductase (FIG. 3, step F); and adihydroxyacetone phosphate reductase (FIG. 3, step G).

In another aspect, provided herein is a non-naturally occurringmicrobial organism, comprising (1) a methanol metabolic pathway, whereinsaid organism comprises at least one exogenous nucleic acid encoding amethanol metabolic pathway enzyme in a sufficient amount to enhance theavailability of reducing equivalents in the presence of methanol; and(2) an glycerol pathway, wherein said organism comprises at least oneexogenous nucleic acid encoding an glycerol pathway enzyme expressed ina sufficient amount to produce glycerol. In one embodiment, the at leastone exogenous nucleic acid encoding the methanol metabolic pathwayenzyme enhances the availability of reducing equivalents in the presenceof methanol in a sufficient amount to increase the amount of glycerolproduced by the non-naturally microbial organism. In some embodiments,the methanol metabolic pathway comprises any of the various combinationsof methanol metabolic pathway enzymes described above or elsewhereherein.

In certain embodiments, (1) the methanol metabolic pathway comprises:1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 1I, 1J, 1K, 1L, 1M, 1N, or 1O or anycombination of 1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 1I, 1J, 1K, 1L, 1M, 1N,or 1O, thereof, wherein 1A is a methanol methyltransferase; 1B is amethylenetetrahydrofolate reductase; 1C is a methylenetetrahydrofolatedehydrogenase; 1D is a methenyltetrahydrofolate cyclohydrolase; 1E is aformyltetrahydrofolate deformylase; 1F is a formyltetrahydrofolatesynthetase; 1G is a formate hydrogen lyase; 1H is a hydrogenase, 1I is aformate dehydrogenase; 1J is a methanol dehydrogenase; 1K is spontaneousor formaldehyde activating enzyme; 1L is a formaldehyde dehydrogenase;1M is spontaneous or a S-(hydroxymethyl)glutathione synthase; 1N isglutathione-dependent formaldehyde dehydrogenase and 1O isS-formylglutathione hydrolase; and (2) the glycerol pathway comprises3A, 3B, 3E, 3F or 3G, or any combination thereof, wherein 3A is aglyceraldehyde-3-phosphate reductase; 3B is a glycerol-3-phosphatephosphatase or a glycerol kinase; 3E is a dihydroxyacetone phosphatephosphatase or a dihydroxyacetone kinase; 3F is a dihydroxyacetonereductase; and 3G is a dihydroxyacetone phosphate reductase. In someembodiments, 1K is spontaneous. In other embodiments, 1K is aformaldehyde activating enzyme. In some embodiments, 1M is spontaneous.In other embodiments, 1M is a S-(hydroxymethyl)glutathione synthase. Insome embodiments, 3B is a glycerol-3-phosphate phosphatase. In otherembodiments, 3C is a glycerol kinase. In some embodiments, 3E is adihydroxyacetone phosphate phosphatase. In other embodiments, 3E is adihydroxyacetone kinase.

In one embodiment, the glycerol pathway comprises 3A. In anotherembodiment, the glycerol pathway comprises 3B. In one embodiment, theglycerol pathway comprises 3E. In another embodiment, the glycerolpathway comprises 3F. In another embodiment, the glycerol pathwaycomprises 3G. In one embodiment, the glycerol pathway comprises 3H. Anycombination of two, three, four or five glycerol pathway enzymes 3A, 3B,3E, 3F and 3G is also contemplated.

In some embodiments, the methanol metabolic pathway is a methanolmetabolic pathway depicted in FIG. 1, and the glycerol pathway is anglycerol pathway depicted in FIG. 3.

Exemplary sets of glycerol pathway enzymes for the conversion of glucoseto glycerol (e.g., via glyceraldehydes-3-phosphate or dihydroxyacetonephosphate) include (i) 3A and 3B; (ii) 3G and 3B; and (iii) 3E and 3F.

In one embodiment, (1) the methanol metabolic pathway comprises 1A and1B; and (2) the glycerol pathway comprises 3A and 3B. In anotherembodiment, (1) the methanol metabolic pathway comprises 1J; and (2) theglycerol pathway comprises 3A and 3B. In one embodiment, (1) themethanol metabolic pathway comprises 1J and 1K; and (2) the glycerolpathway comprises 3A and 3B. In certain embodiments, (1) the methanolmetabolic pathway comprises 1A, 1B, 1C, 1D, and 1E; and (2) the glycerolpathway comprises 3A and 3B. In some embodiments, (1) the methanolmetabolic pathway comprises 1A, 1B, 1C, 1D and 1F; and (2) the glycerolpathway comprises 3A and 3B. In some embodiments, (1) the methanolmetabolic pathway comprises 1J, 1C, 1D and 1E; and (2) the glycerolpathway comprises 3A and 3B. In one embodiment, (1) the methanolmetabolic pathway comprises 1J, 1C, 1D and 1F; and (2) the glycerolpathway comprises 3A and 3B. In another embodiment, (1) the methanolmetabolic pathway comprises 1J and 1L; and (2) the glycerol pathwaycomprises 3A and 3B. In yet another embodiment, (1) the methanolmetabolic pathway comprises 1J, 1M, 1N and 1O; and (2) the glycerolpathway comprises 3A and 3B. In certain embodiments, (1) the methanolmetabolic pathway comprises 1J, 1N and 1O; and (2) the glycerol pathwaycomprises 3A and 3B. In some embodiments, (1) the methanol metabolicpathway comprises 1J, 1K, 1C, 1D and 1E; and (2) the glycerol pathwaycomprises 3A and 3B. In one embodiment, (1) the methanol metabolicpathway comprises 1J, 1K, 1C, 1D and 1F; and (2) the glycerol pathwaycomprises 3A and 3B. In certain embodiments, (1) the methanol metabolicpathway comprises 1I; and (2) the glycerol pathway comprises 3A and 3B.In certain embodiments, (1) the methanol metabolic pathway comprises 1A,1B, 1C, 1D, 1E and 1I; and (2) the glycerol pathway comprises 3A and 3B.In some embodiments, (1) the methanol metabolic pathway comprises 1A,1B, 1C, 1D, 1F and 1I; and (2) the glycerol pathway comprises 3A and 3B.In some embodiments, (1) the methanol metabolic pathway comprises 1J,1C, 1D, 1E and 1I; and (2) the glycerol pathway comprises 3A and 3B. Inone embodiment, (1) the methanol metabolic pathway comprises 1J, 1C, 1D,1F and 1I; and (2) the glycerol pathway comprises 3A and 3B. In anotherembodiment, (1) the methanol metabolic pathway comprises 1J, 1L and 1I;and (2) the glycerol pathway comprises 3A and 3B. In yet anotherembodiment, (1) the methanol metabolic pathway comprises 1J, 1M, 1N, 1Oand 1I; and (2) the glycerol pathway comprises 3A and 3B. In certainembodiments, (1) the methanol metabolic pathway comprises 1J, 1N, 1O and1I; and (2) the glycerol pathway comprises 3A and 3B. In someembodiments, (1) the methanol metabolic pathway comprises 1J, 1K, 1C,1D, 1E and 1I; and (2) the glycerol pathway comprises 3A and 3B. In oneembodiment, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D,1F and 1I; and (2) the glycerol pathway comprises 3A and 3B. In certainembodiments, (1) the methanol metabolic pathway comprises 1G; and (2)the glycerol pathway comprises 3A and 3B. In certain embodiments, (1)the methanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1E and 1G; and(2) the glycerol pathway comprises 3A and 3B. In some embodiments, (1)the methanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1F and 1G; and(2) the glycerol pathway comprises 3A and 3B. In some embodiments, (1)the methanol metabolic pathway comprises 1J, 1C, 1D, 1E and 1G; and (2)the glycerol pathway comprises 3A and 3B. In one embodiment, (1) themethanol metabolic pathway comprises 1J, 1C, 1D, 1F and 1G; and (2) theglycerol pathway comprises 3A and 3B. In another embodiment, (1) themethanol metabolic pathway comprises 1J, 1L and 1G; and (2) the glycerolpathway comprises 3A and 3B. In yet another embodiment, (1) the methanolmetabolic pathway comprises 1J, 1M, 1N, 1O and 1G; and (2) the glycerolpathway comprises 3A and 3B. In certain embodiments, (1) the methanolmetabolic pathway comprises 1J, 1N, 1O and 1G; and (2) the glycerolpathway comprises 3A and 3B. In some embodiments, (1) the methanolmetabolic pathway comprises 1J, 1K, 1C, 1D, 1E and 1G; and (2) theglycerol pathway comprises 3A and 3B. In one embodiment, (1) themethanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1F and 1G; and (2)the glycerol pathway comprises 3A and 3B. In certain embodiments, (1)the methanol metabolic pathway comprises 1G and 1H; and (2) the glycerolpathway comprises 3A and 3B. In certain embodiments, (1) the methanolmetabolic pathway comprises 1A, 1B, 1C, 1D, 1E, 1G and 1H; and (2) theglycerol pathway comprises 3A and 3B. In some embodiments, (1) themethanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1F, 1G and 1H; and(2) the glycerol pathway comprises 3A and 3B. In some embodiments, (1)the methanol metabolic pathway comprises 1J, 1C, 1D, 1E, 1G and 1H; and(2) the glycerol pathway comprises 3A and 3B. In one embodiment, (1) themethanol metabolic pathway comprises 1J, 1C, 1D, 1F, 1G and 1H; and (2)the glycerol pathway comprises 3A and 3B. In another embodiment, (1) themethanol metabolic pathway comprises 1J, 1L, 1G and 1H; and (2) theglycerol pathway comprises 3A and 3B. In yet another embodiment, (1) themethanol metabolic pathway comprises 1J, 1M, 1N, 1O, 1G and 1H; and (2)the glycerol pathway comprises 3A and 3B. In certain embodiments, (1)the methanol metabolic pathway comprises 1J, 1N, 1O, 1G and 1H; and (2)the glycerol pathway comprises 3A and 3B. In some embodiments, (1) themethanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1E, 1G and 1H; and(2) the glycerol pathway comprises 3A and 3B. In one embodiment, (1) themethanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1F, 1G and 1H; and(2) the glycerol pathway comprises 3A and 3B. In some embodiments, 1K isspontaneous. In other embodiments, 1K is a formaldehyde activatingenzyme. In some embodiments, 1M is spontaneous. In other embodiments, 1Mis a S-(hydroxymethyl)glutathione synthase. In some embodiments, 3B is aglycerol-3-phosphate phosphatase. In other embodiments, 3C is a glycerolkinase. In some embodiments, 3E is a dihydroxyacetone phosphatephosphatase. In other embodiments, 3E is a dihydroxyacetone kinase.

In one embodiment, (1) the methanol metabolic pathway comprises 1A and1B; and (2) the glycerol pathway comprises 3G and 3B. In anotherembodiment, (1) the methanol metabolic pathway comprises 1J; and (2) theglycerol pathway comprises 3G and 3B. In one embodiment, (1) themethanol metabolic pathway comprises 1J and 1K; and (2) the glycerolpathway comprises 3G and 3B. In certain embodiments, (1) the methanolmetabolic pathway comprises 1A, 1B, 1C, 1D, and 1E; and (2) the glycerolpathway comprises 3G and 3B. In some embodiments, (1) the methanolmetabolic pathway comprises 1A, 1B, 1C, 1D and 1F; and (2) the glycerolpathway comprises 3G and 3B. In some embodiments, (1) the methanolmetabolic pathway comprises 1J, 1C, 1D and 1E; and (2) the glycerolpathway comprises 3G and 3B. In one embodiment, (1) the methanolmetabolic pathway comprises 1J, 1C, 1D and 1F; and (2) the glycerolpathway comprises 3G and 3B. In another embodiment, (1) the methanolmetabolic pathway comprises 1J and 1L; and (2) the glycerol pathwaycomprises 3G and 3B. In yet another embodiment, (1) the methanolmetabolic pathway comprises 1J, 1M, 1N and 1O; and (2) the glycerolpathway comprises 3G and 3B. In certain embodiments, (1) the methanolmetabolic pathway comprises 1J, 1N and 1O; and (2) the glycerol pathwaycomprises 3G and 3B. In some embodiments, (1) the methanol metabolicpathway comprises 1J, 1K, 1C, 1D and 1E; and (2) the glycerol pathwaycomprises 3G and 3B. In one embodiment, (1) the methanol metabolicpathway comprises 1J, 1K, 1C, 1D and 1F; and (2) the glycerol pathwaycomprises 3G and 3B. In certain embodiments, (1) the methanol metabolicpathway comprises 1I; and (2) the glycerol pathway comprises 3G and 3B.In certain embodiments, (1) the methanol metabolic pathway comprises 1A,1B, 1C, 1D, 1E and 1I; and (2) the glycerol pathway comprises 3G and 3B.In some embodiments, (1) the methanol metabolic pathway comprises 1A,1B, 1C, 1D, 1F and 1I; and (2) the glycerol pathway comprises 3G and 3B.In some embodiments, (1) the methanol metabolic pathway comprises 1J,1C, 1D, 1E and 1I; and (2) the glycerol pathway comprises 3G and 3B. Inone embodiment, (1) the methanol metabolic pathway comprises 1J, 1C, 1D,1F and 1I; and (2) the glycerol pathway comprises 3G and 3B. In anotherembodiment, (1) the methanol metabolic pathway comprises 1J, 1L and 1I;and (2) the glycerol pathway comprises 3G and 3B. In yet anotherembodiment, (1) the methanol metabolic pathway comprises 1J, 1M, 1N, 1Oand 1I; and (2) the glycerol pathway comprises 3G and 3B. In certainembodiments, (1) the methanol metabolic pathway comprises 1J, 1N, 1O and1I; and (2) the glycerol pathway comprises 3G and 3B. In someembodiments, (1) the methanol metabolic pathway comprises 1J, 1K, 1C,1D, 1E and 1I; and (2) the glycerol pathway comprises 3G and 3B. In oneembodiment, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D,1F and 1I; and (2) the glycerol pathway comprises 3G and 3B. In certainembodiments, (1) the methanol metabolic pathway comprises 1G; and (2)the glycerol pathway comprises 3G and 3B. In certain embodiments, (1)the methanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1E and 1G; and(2) the glycerol pathway comprises 3G and 3B. In some embodiments, (1)the methanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1F and 1G; and(2) the glycerol pathway comprises 3G and 3B. In some embodiments, (1)the methanol metabolic pathway comprises 1J, 1C, 1D, 1E and 1G; and (2)the glycerol pathway comprises 3G and 3B. In one embodiment, (1) themethanol metabolic pathway comprises 1J, 1C, 1D, 1F and 1G; and (2) theglycerol pathway comprises 3G and 3B. In another embodiment, (1) themethanol metabolic pathway comprises 1J, 1L and 1G; and (2) the glycerolpathway comprises 3G and 3B. In yet another embodiment, (1) the methanolmetabolic pathway comprises 1J, 1M, 1N, 1O and 1G; and (2) the glycerolpathway comprises 3G and 3B. In certain embodiments, (1) the methanolmetabolic pathway comprises 1J, 1N, 1O and 1G; and (2) the glycerolpathway comprises 3G and 3B. In some embodiments, (1) the methanolmetabolic pathway comprises 1J, 1K, 1C, 1D, 1E and 1G; and (2) theglycerol pathway comprises 3G and 3B. In one embodiment, (1) themethanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1F and 1G; and (2)the glycerol pathway comprises 3G and 3B. In certain embodiments, (1)the methanol metabolic pathway comprises 1G and 1H; and (2) the glycerolpathway comprises 3G and 3B. In certain embodiments, (1) the methanolmetabolic pathway comprises 1A, 1B, 1C, 1D, 1E, 1G and 1H; and (2) theglycerol pathway comprises 3G and 3B. In some embodiments, (1) themethanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1F, 1G and 1H; and(2) the glycerol pathway comprises 3G and 3B. In some embodiments, (1)the methanol metabolic pathway comprises 1J, 1C, 1D, 1E, 1G and 1H; and(2) the glycerol pathway comprises 3G and 3B. In one embodiment, (1) themethanol metabolic pathway comprises 1J, 1C, 1D, 1F, 1G and 1H; and (2)the glycerol pathway comprises 3G and 3B. In another embodiment, (1) themethanol metabolic pathway comprises 1J, 1L, 1G and 1H; and (2) theglycerol pathway comprises 3G and 3B. In yet another embodiment, (1) themethanol metabolic pathway comprises 1J, 1M, 1N, 1O, 1G and 1H; and (2)the glycerol pathway comprises 3G and 3B. In certain embodiments, (1)the methanol metabolic pathway comprises 1J, 1N, 1O, 1G and 1H; and (2)the glycerol pathway comprises 3G and 3B. In some embodiments, (1) themethanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1E, 1G and 1H; and(2) the glycerol pathway comprises 3G and 3B. In one embodiment, (1) themethanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1F, 1G and 1H; and(2) the glycerol pathway comprises 3G and 3B. In some embodiments, 1K isspontaneous. In other embodiments, 1K is a formaldehyde activatingenzyme. In some embodiments, 1M is spontaneous. In other embodiments, 1Mis a S-(hydroxymethyl)glutathione synthase. In some embodiments, 3B is aglycerol-3-phosphate phosphatase. In other embodiments, 3C is a glycerolkinase. In some embodiments, 3E is a dihydroxyacetone phosphatephosphatase. In other embodiments, 3E is a dihydroxyacetone kinase.

In one embodiment, (1) the methanol metabolic pathway comprises 1A and1B; and (2) the glycerol pathway comprises 3E and 3F. In anotherembodiment, (1) the methanol metabolic pathway comprises 1J; and (2) theglycerol pathway comprises 3E and 3F. In one embodiment, (1) themethanol metabolic pathway comprises 1J and 1K; and (2) the glycerolpathway comprises 3E and 3F. In certain embodiments, (1) the methanolmetabolic pathway comprises 1A, 1B, 1C, 1D, and 1E; and (2) the glycerolpathway comprises 3E and 3F. In some embodiments, (1) the methanolmetabolic pathway comprises 1A, 1B, 1C, 1D and 1F; and (2) the glycerolpathway comprises 3E and 3F. In some embodiments, (1) the methanolmetabolic pathway comprises 1J, 1C, 1D and 1E; and (2) the glycerolpathway comprises 3E and 3F. In one embodiment, (1) the methanolmetabolic pathway comprises 1J, 1C, 1D and 1F; and (2) the glycerolpathway comprises 3E and 3F. In another embodiment, (1) the methanolmetabolic pathway comprises 1J and 1L; and (2) the glycerol pathwaycomprises 3E and 3F. In yet another embodiment, (1) the methanolmetabolic pathway comprises 1J, 1M, 1N and 1O; and (2) the glycerolpathway comprises 3E and 3F. In certain embodiments, (1) the methanolmetabolic pathway comprises 1J, 1N and 1O; and (2) the glycerol pathwaycomprises 3E and 3F. In some embodiments, (1) the methanol metabolicpathway comprises 1J, 1K, 1C, 1D and 1E; and (2) the glycerol pathwaycomprises 3E and 3F. In one embodiment, (1) the methanol metabolicpathway comprises 1J, 1K, 1C, 1D and 1F; and (2) the glycerol pathwaycomprises 3E and 3F. In certain embodiments, (1) the methanol metabolicpathway comprises 1I; and (2) the glycerol pathway comprises 3E and 3F.In certain embodiments, (1) the methanol metabolic pathway comprises 1A,1B, 1C, 1D, 1E and 1I; and (2) the glycerol pathway comprises 3E and 3F.In some embodiments, (1) the methanol metabolic pathway comprises 1A,1B, 1C, 1D, 1F and 1I; and (2) the glycerol pathway comprises 3E and 3F.In some embodiments, (1) the methanol metabolic pathway comprises 1J,1C, 1D, 1E and 1I; and (2) the glycerol pathway comprises 3E and 3F. Inone embodiment, (1) the methanol metabolic pathway comprises 1J, 1C, 1D,1F and 1I; and (2) the glycerol pathway comprises 3E and 3F. In anotherembodiment, (1) the methanol metabolic pathway comprises 1J, 1L and 1I;and (2) the glycerol pathway comprises 3E and 3F. In yet anotherembodiment, (1) the methanol metabolic pathway comprises 1J, 1M, 1N, 1Oand 1I; and (2) the glycerol pathway comprises 3E and 3F. In certainembodiments, (1) the methanol metabolic pathway comprises 1J, 1N, 1O and1I; and (2) the glycerol pathway comprises 3E and 3F. In someembodiments, (1) the methanol metabolic pathway comprises 1J, 1K, 1C,1D, 1E and 1I; and (2) the glycerol pathway comprises 3E and 3F. In oneembodiment, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D,1F and 1I; and (2) the glycerol pathway comprises 3E and 3F. In certainembodiments, (1) the methanol metabolic pathway comprises 1G; and (2)the glycerol pathway comprises 3E and 3F. In certain embodiments, (1)the methanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1E and 1G; and(2) the glycerol pathway comprises 3E and 3F. In some embodiments, (1)the methanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1F and 1G; and(2) the glycerol pathway comprises 3E and 3F. In some embodiments, (1)the methanol metabolic pathway comprises 1J, 1C, 1D, 1E and 1G; and (2)the glycerol pathway comprises 3E and 3F. In one embodiment, (1) themethanol metabolic pathway comprises 1J, 1C, 1D, 1F and 1G; and (2) theglycerol pathway comprises 3E and 3F. In another embodiment, (1) themethanol metabolic pathway comprises 1J, 1L and 1G; and (2) the glycerolpathway comprises 3E and 3F. In yet another embodiment, (1) the methanolmetabolic pathway comprises 1J, 1M, 1N, 1O and 1G; and (2) the glycerolpathway comprises 3E and 3F. In certain embodiments, (1) the methanolmetabolic pathway comprises 1J, 1N, 1O and 1G; and (2) the glycerolpathway comprises 3E and 3F. In some embodiments, (1) the methanolmetabolic pathway comprises 1J, 1K, 1C, 1D, 1E and 1G; and (2) theglycerol pathway comprises 3E and 3F. In one embodiment, (1) themethanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1F and 1G; and (2)the glycerol pathway comprises 3E and 3F. In certain embodiments, (1)the methanol metabolic pathway comprises 1G and 1H; and (2) the glycerolpathway comprises 3E and 3F. In certain embodiments, (1) the methanolmetabolic pathway comprises 1A, 1B, 1C, 1D, 1E, 1G and 1H; and (2) theglycerol pathway comprises 3E and 3F. In some embodiments, (1) themethanol metabolic pathway comprises 1A, 1B, 1C, 1D, 1F, 1G and 1H; and(2) the glycerol pathway comprises 3E and 3F. In some embodiments, (1)the methanol metabolic pathway comprises 1J, 1C, 1D, 1E, 1G and 1H; and(2) the glycerol pathway comprises 3E and 3F. In one embodiment, (1) themethanol metabolic pathway comprises 1J, 1C, 1D, 1F, 1G and 1H; and (2)the glycerol pathway comprises 3E and 3F. In another embodiment, (1) themethanol metabolic pathway comprises 1J, 1L, 1O and 1H; and (2) theglycerol pathway comprises 3E and 3F. In yet another embodiment, (1) themethanol metabolic pathway comprises 1J, 1M, 1N, 1O, 1G and 1H; and (2)the glycerol pathway comprises 3E and 3F. In certain embodiments, (1)the methanol metabolic pathway comprises 1J, 1N, 1O, 1G and 1H; and (2)the glycerol pathway comprises 3E and 3F. In some embodiments, (1) themethanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1E, 1G and 1H; and(2) the glycerol pathway comprises 3E and 3F. In one embodiment, (1) themethanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1F, 10 and 1H; and(2) the glycerol pathway comprises 3E and 3F. In some embodiments, 1K isspontaneous. In other embodiments, 1K is a formaldehyde activatingenzyme. In some embodiments, 1M is spontaneous. In other embodiments, 1Mis a S-(hydroxymethyl)glutathione synthase. In some embodiments, 3B is aglycerol-3-phosphate phosphatase. In other embodiments, 3C is a glycerolkinase. In some embodiments, 3E is a dihydroxyacetone phosphatephosphatase. In other embodiments, 3E is a dihydroxyacetone kinase.

In one embodiment, the non-naturally occurring microbial organismcomprises (1) a methanol metabolic pathway comprising 1A and 1B; 1J; 1Jand 1K; 1A, 1B, 1C, 1D, and 1E; 1A, 1B, 1C, 1D and 1F; 1J, 1C, 1D and1E; 1J, 1C, 1D and 1F; 1J and 1L; 1J, 1M, 1N and 1O; 1J, 1N and 1O; 1J,1K, 1C, 1D and 1E; 1J, 1K, 1C, 1D and 1F; 1I; 1A, 1B, 1C, 1D, 1E and 1I;1A, 1B, 1C, 1D, 1F and 1I; 1J, 1C, 1D, 1E and 1I; 1J, 1C, 1D, 1F and 1I;1J, 1L and 1I; 1J, 1M, 1N, 1O and 1I; 1J, 1N, 1O and 1I; 1J, 1K, 1C, 1D,1E and 1I; 1J, 1K, 1C, 1D, 1F and 1I; 1G; 1A, 1B, 1C, 1D, 1E and 1G; 1A,1B, 1C, 1D, 1F and 1G; 1J, 1C, 1D, 1E and 1G; 1J, 1C, 1D, 1F and 1G; 1J,1L and 1O; 1J, 1M, 1N, 1O and 1G; 1J, 1N, 1O and 1G; 1J, 1K, 1C, 1D, 1Eand 1G; 1J, 1K, 1C, 1D, 1F and 1G; 1G and 1H; 1A, 1B, 1C, 1D, 1E, 1G and1H; 1A, 1B, 1C, 1D, 1F, 1G and 1H; 1J, 1C, 1D, 1E, 1G and 1H; 1J, 1C,1D, 1F, 1G and 1H; 1J, 1L, 1G and 1H; 1J, 1M, 1N, 1O, 1G and 1H; 1J, 1N,1O, 1G and 1H; 1J, 1K, 1C, 1D, 1E, 1G and 1H; or 1J, 1K, 1C, 1D, 1F, 1Gand 1H; and (2) a glycerol pathway. In some embodiments, 1K isspontaneous. In other embodiments, 1K is a formaldehyde activatingenzyme. In some embodiments, 1M is spontaneous. In other embodiments, 1Mis a S-(hydroxymethyl)glutathione synthase.

Any methanol metabolic pathway provided herein can be combined with anyglycerol pathway provided herein.

Also provided herein are exemplary pathways, which utilize formaldehydeproduced from the oxidation of methanol (e.g., as provided in FIG. 1,step J) in the formation of intermediates of certain central metabolicpathways that can be used for the formation of biomass. One exemplaryformaldehyde assimilation pathway that can utilize formaldehyde producedfrom the oxidation of methanol (e.g., as provided in FIG. 1) is shown inFIG. 4, which involves condensation of formaldehyde andD-ribulose-5-phosphate to form hexylose-6-phosphate (h6p) byhexylose-6-phosphate synthase (FIG. 4, step A). The enzyme can use Mg²⁺or Mn²⁺ for maximal activity, although other metal ions are useful, andeven non-metal-ion-dependent mechanisms are contemplated. H6p isconverted into fructose-6-phosphate by 6-phospho-3-hexyloisomerase (FIG.4, step B). Another exemplary pathway that involves the detoxificationand assimilation of formaldehyde produced from the oxidation of methanol(e.g., as provided in FIG. 1) is shown in FIG. 5 and proceeds throughdihydroxyacetone. Dihydroxyacetone synthase is a special transketolasethat first transfers a glycoaldehyde group from xylulose-5-phosphate toformaldehyde, resulting in the formulation of dihydroxyacetone (DHA) andglyceraldehyde-3-phosphate (G3P), which is an intermediate in glycolysis(FIG. 5, step A). The DHA obtained from DHA synthase is then furtherphosphorylated to form DHA phosphate by a DHA kinase (FIG. 5, step B).DHAP can be assimilated into glycolysis and several other pathways.Rather than converting formaldehyde to formate and on to CO₂ off-gassed,the pathways provided in FIGS. 4 and 5 show that carbon is assimilated,going into the final product.

Thus, in one embodiment, an organism having a methanol metabolicpathway, either alone or in combination with a 1,2-propanediol,n-propanol, 1,3-propanediol or glycerol pathway, as provided herein,further comprises a formaldehyde assimilation pathway that utilizesformaldehyde, e.g., obtained from the oxidation of methanol, in theformation of intermediates of certain central metabolic pathways thatcan be used, for example, in the formation of biomass. In some ofembodiments, the formaldehyde assimilation pathway comprises 4A or 4B,wherein 4A is a hexylose-6-phosphate synthase and 4B is a6-phospho-3-hexyloisomerase In other embodiments, the formaldehydeassimilation pathway comprises 5A or 5B, wherein 5A is adihydroxyacetone synthase and 5B is a dihydroxyacetone kinase.

In certain embodiments, provided herein is a non-naturally occurringmicrobial organism having a methanol metabolic pathway, wherein saidorganism comprises at least one exogenous nucleic acid encoding amethanol dehydrogenase (1J) expressed in a sufficient amount to enhancethe availability of reducing equivalents in the presence of methanoland/or expressed in a sufficient amount to convert methanol toformaldehyde. In some embodiments, the microbial organism furthercomprises a formaldehyde assimilation pathway. In certain embodiments,the organism further comprises at least one exogenous nucleic acidencoding a formaldehyde assimilation pathway enzyme expressed in asufficient amount to produce an intermediate of glycolysis and/or ametabolic pathway that can be used, for example, in the formation ofbiomass. In certain embodiments, the formaldehyde assimilation pathwayenzyme is selected from the group consisting of a hexylose-6-phosphatesynthase (4A), 6-phospho-3-hexyloisomerase (4B), dihydroxyacetonesynthase (5A) and dihydroxyacetone kinase (5B).

In one aspect, provided herein is a non-naturally occurring microbialorganism, comprising (1) a methanol metabolic pathway, wherein saidorganism comprises at least one exogenous nucleic acid encoding amethanol metabolic pathway enzyme in a sufficient amount to enhance theavailability of reducing equivalents in the presence of methanol and/orexpressed in a sufficient amount to convert methanol to formaldehyde;and (2) a formaldehyde assimilation pathway, wherein said organismcomprises at least one exogenous nucleic acid encoding a formaldehydeassimilation pathway enzyme expressed in a sufficient amount to producean intermediate of glycolysis and/or a metabolic pathway that can beused, for example, in the formation of biomass. In specific embodiments,the methanol metabolic pathway comprises a methanol dehydrogenase (1J).In certain embodiments, the formaldehyde assimilation pathway enzyme is4A, and the intermediate is a hexylose-6-phosphate, afructose-6-phosphate, or a combination thereof. In other embodiments,the formaldehyde assimilation pathway enzyme is 4B, and the intermediateis a hexylose-6-phosphate, a fructose-6-phosphate, or a combinationthereof. In yet other embodiments, the formaldehyde assimilation pathwayenzyme is 4A and 4B, and the intermediate is a hexylose-6-phosphate, afructose-6-phosphate, or a combination thereof. In some embodiments, theformaldehyde assimilation pathway enzyme is 5A, and the intermediate isa dihydroxyacetone (DHA), a dihydroxyacetone phosphate, or a combinationthereof. In other embodiments, the formaldehyde assimilation pathwayenzyme is 5B, and the intermediate is a DHA, a dihydroxyacetonephosphate, or a combination thereof. In yet other embodiments, theformaldehyde assimilation pathway enzyme is 5A and 5B, and theintermediate is a DHA, a dihydroxyacetone phosphate, or a combinationthereof. In one embodiment, the at least one exogenous nucleic acidencoding the methanol metabolic pathway enzyme, in the presence ofmethanol, sufficiently enhances the availability of reducing equivalentsand sufficiently increases formaldehyde assimilation to increase theproduction of 1,2-propanediol, n-propanol, 1,3-propanediol, glycerol, orother products described herein by the non-naturally microbial organism.In some embodiments, the methanol metabolic pathway comprises any of thevarious combinations of methanol metabolic pathway enzymes describedabove or elsewhere herein.

In certain embodiments, (1) the methanol metabolic pathway comprises:1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 1I, 1J, 1K, 1L, 1M, 1N, or 1O or anycombination of 1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 1I, 1J, 1K, 1L, 1M, 1N,or 1O, thereof, wherein 1A is a methanol methyltransferase; 1B is amethylenetetrahydrofolate reductase; 1C is a methylenetetrahydrofolatedehydrogenase; 1D is a methenyltetrahydrofolate cyclohydrolase; 1E is aformyltetrahydrofolate deformylase; 1F is a formyltetrahydrofolatesynthetase; 1G is a formate hydrogen lyase; 1H is a hydrogenase, 1I is aformate dehydrogenase; 1J is a methanol dehydrogenase; 1K is spontaneousor formaldehyde activating enzyme; 1L is a formaldehyde dehydrogenase;1M is spontaneous or a S-(hydroxymethyl)glutathione synthase; 1N isglutathione-dependent formaldehyde dehydrogenase and 1O isS-formylglutathione hydrolase; and (2) the formaldehyde assimilationpathway comprises 4A, 4B or a combination thereof, wherein 4A is ahexylose-6-phosphate synthase, and 4B is a 6-phospho-3-hexyloisomerase.In some embodiments, 1K is spontaneous. In other embodiments, 1K is aformaldehyde activating enzyme. In some embodiments, 1M is spontaneous.In other embodiments, 1M is a S-(hydroxymethyl)glutathione synthase. Insome embodiments, the intermediate is a hexylose-6-phosphate. In otherembodiments, the intermediate is a fructose-6-phosphate. In yet otherembodiments, the intermediate is a hexylose-6-phosphate and afructose-6-phosphate.

In one embodiment, the formaldehyde assimilation pathway comprises 4A.In another embodiment, the formaldehyde assimilation pathway comprises4B. In one embodiment, the formaldehyde assimilation pathway comprises4A and 4B.

In some embodiments, the methanol metabolic pathway is a methanolmetabolic pathway depicted in FIG. 1, and a formaldehyde assimilationpathway depicted in FIG. 4. An exemplary set of formaldehydeassimilation pathway enzymes to convert D-ribulose-5-phosphate andformaldehyde to fructose-6-phosphate (via hexylose-6-phosphate)according to FIG. 4 include 4A and 4B.

In a specific embodiment, (1) the methanol metabolic pathway comprises1J; and (2) the formaldehyde assimilation pathway comprises 4A and 4B.In other embodiments, (1) the methanol metabolic pathway comprises 1Jand 1K; and (2) the formaldehyde assimilation pathway comprises 4A and4B. In some embodiments, (1) the methanol metabolic pathway comprises1J, 1C, 1D and 1E; and (2) the formaldehyde assimilation pathwaycomprises 4A and 4B. In one embodiment, (1) the methanol metabolicpathway comprises 1J, 1C, 1D and 1F; and (2) the formaldehydeassimilation pathway comprises 4A and 4B. In another embodiment, (1) themethanol metabolic pathway comprises 1J and 1L; and (2) the formaldehydeassimilation pathway comprises 4A and 4B. In yet another embodiment, (1)the methanol metabolic pathway comprises 1J, 1M, 1N and 1O; and (2) theformaldehyde assimilation pathway comprises 4A and 4B. In certainembodiments, (1) the methanol metabolic pathway comprises 1J, 1N and 1O;and (2) the formaldehyde assimilation pathway comprises 4A and 4B. Insome embodiments, (1) the methanol metabolic pathway comprises 1J, 1K,1C, 1D and 1E; and (2) the formaldehyde assimilation pathway comprises4A and 4B. In one embodiment, (1) the methanol metabolic pathwaycomprises 1J, 1K, 1C, 1D and 1F; and (2) the formaldehyde assimilationpathway comprises 4A and 4B. In some embodiments, (1) the methanolmetabolic pathway comprises 1J, 1C, 1D, 1E and 1I; and (2) theformaldehyde assimilation pathway comprises 4A and 4B. In oneembodiment, (1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1Fand 1I; and (2) the formaldehyde assimilation pathway comprises 4A and4B. In another embodiment, (1) the methanol metabolic pathway comprises1J, 1L and 1I; and (2) the formaldehyde assimilation pathway comprises4A and 4B. In yet another embodiment, (1) the methanol metabolic pathwaycomprises 1J, 1M, 1N, 1O and 1I; and (2) the formaldehyde assimilationpathway comprises 4A and 4B. In certain embodiments, (1) the methanolmetabolic pathway comprises 1J, 1N, 1O and 1I; and (2) the formaldehydeassimilation pathway comprises 4A and 4B. In some embodiments, (1) themethanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1E and 1I; and (2)the formaldehyde assimilation pathway comprises 4A and 4B. In oneembodiment, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D,1F and 1I; and (2) the formaldehyde assimilation pathway comprises 4Aand 4B. In some embodiments, (1) the methanol metabolic pathwaycomprises 1J, 1C, 1D, 1E and 1G; and (2) the formaldehyde assimilationpathway comprises 4A and 4B. In one embodiment, (1) the methanolmetabolic pathway comprises 1J, 1C, 1D, 1F and 1G; and (2) theformaldehyde assimilation pathway comprises 4A and 4B. In anotherembodiment, (1) the methanol metabolic pathway comprises 1J, 1L and 1G;and (2) the formaldehyde assimilation pathway comprises 4A and 4B. Inyet another embodiment, (1) the methanol metabolic pathway comprises 1J,1M, 1N, 1O and 1G; and (2) the formaldehyde assimilation pathwaycomprises 4A and 4B. In certain embodiments, (1) the methanol metabolicpathway comprises 1J, 1N, 1O and 1G; and (2) the formaldehydeassimilation pathway comprises 4A and 4B. In some embodiments, (1) themethanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1E and 1G; and (2)the formaldehyde assimilation pathway comprises 4A and 4B. In oneembodiment, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D,1F and 1G; and (2) the formaldehyde assimilation pathway comprises 4Aand 4B. In some embodiments, (1) the methanol metabolic pathwaycomprises 1J, 1C, 1D, 1E, 1G and 1H; and (2) the formaldehydeassimilation pathway comprises 4A and 4B. In one embodiment, (1) themethanol metabolic pathway comprises 1J, 1C, 1D, 1F, 1G and 1H; and (2)the formaldehyde assimilation pathway comprises 4A and 4B. In anotherembodiment, (1) the methanol metabolic pathway comprises 1J, 1L, 1G and1H; and (2) the formaldehyde assimilation pathway comprises 4A and 4B.In yet another embodiment, (1) the methanol metabolic pathway comprises1J, 1M, 1N, 1O, 1G and 1H; and (2) the formaldehyde assimilation pathwaycomprises 4A and 4B. In certain embodiments, (1) the methanol metabolicpathway comprises 1J, 1N, 1O, 1G and 1H; and (2) the formaldehydeassimilation pathway comprises 4A and 4B. In some embodiments, (1) themethanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1E, 1G and 1H; and(2) the formaldehyde assimilation pathway comprises 4A and 4B. In oneembodiment, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D,1F, 1G and 1H; and (2) the formaldehyde assimilation pathway comprises4A and 4B. In some embodiments, 1K is spontaneous. In other embodiments,1K is a formaldehyde activating enzyme. In some embodiments, 1M isspontaneous. In some embodiments, the intermediate is ahexylose-6-phosphate. In other embodiments, the intermediate is afructose-6-phosphate. In yet other embodiments, the intermediate is ahexylose-6-phosphate and a fructose-6-phosphate.

In certain embodiments, (1) the methanol metabolic pathway comprises:1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 1I, 1J, 1K, 1L, 1M, 1N, or 1O or anycombination of 1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 1I, 1J, 1K, 1L, 1M, 1N,or 1O, thereof, wherein 1A is a methanol methyltransferase; 1B is amethylenetetrahydrofolate reductase; 1C is a methylenetetrahydrofolatedehydrogenase; 1D is a methenyltetrahydrofolate cyclohydrolase; 1E is aformyltetrahydrofolate deformylase; 1F is a formyltetrahydrofolatesynthetase; 1G is a formate hydrogen lyase; 1H is a hydrogenase, 1I is aformate dehydrogenase; 1J is a methanol dehydrogenase; 1K is spontaneousor formaldehyde activating enzyme; 1L is a formaldehyde dehydrogenase;1M is spontaneous or a S-(hydroxymethyl)glutathione synthase; 1N isglutathione-dependent formaldehyde dehydrogenase and 1O isS-formylglutathione hydrolase; and (2) the formaldehyde assimilationpathway comprises 5A, 5B or a combination thereof, wherein 5A is adihydroxyacetone synthase and 5B is a dihydroxyacetone kinase. In someembodiments, 1K is spontaneous. In other embodiments, 1K is aformaldehyde activating enzyme. In some embodiments, 1M is spontaneous.In other embodiments, 1M is a S-(hydroxymethyl)glutathione synthase. Insome embodiments, the intermediate is a DHA. In other embodiments, theintermediate is a dihydroxyacetone phosphate. In yet other embodiments,the intermediate is a DHA and a dihydroxyacetone phosphate.

In one embodiment, the formaldehyde assimilation pathway comprises 5A.In another embodiment, the formaldehyde assimilation pathway comprises5B. In one embodiment, the formaldehyde assimilation pathway comprises5A and 5B.

In some embodiments, the methanol metabolic pathway is a methanolmetabolic pathway depicted in FIG. 1, and a formaldehyde assimilationpathway depicted in FIG. 5. An exemplary set of formaldehydeassimilation pathway enzymes to convert xyulose-5-phosphate andformaldehyde to dihydroxyacetone-phosphate (via DHA) according to FIG. 5include 5A and 5B.

In a specific embodiment, (1) the methanol metabolic pathway comprises1J; and (2) the formaldehyde assimilation pathway comprises 5A and 5B.In other embodiments, (1) the methanol metabolic pathway comprises 1Jand 1K; and (2) the formaldehyde assimilation pathway comprises 5A and5B. In some embodiments, (1) the methanol metabolic pathway comprises1J, 1C, 1D and 1E; and (2) the formaldehyde assimilation pathwaycomprises 5A and 5B. In one embodiment, (1) the methanol metabolicpathway comprises 1J, 1C, 1D and 1F; and (2) the formaldehydeassimilation pathway comprises 5A and 5B. In another embodiment, (1) themethanol metabolic pathway comprises 1J and 1L; and (2) the formaldehydeassimilation pathway comprises 5A and 5B. In yet another embodiment, (1)the methanol metabolic pathway comprises 1J, 1M, 1N and 1O; and (2) theformaldehyde assimilation pathway comprises 5A and 5B. In certainembodiments, (1) the methanol metabolic pathway comprises 1J, 1N and 1O;and (2) the formaldehyde assimilation pathway comprises 5A and 5B. Insome embodiments, (1) the methanol metabolic pathway comprises 1J, 1K,1C, 1D and 1E; and (2) the formaldehyde assimilation pathway comprises5A and 5B. In one embodiment, (1) the methanol metabolic pathwaycomprises 1J, 1K, 1C, 1D and 1F; and (2) the formaldehyde assimilationpathway comprises 5A and 5B. In some embodiments, (1) the methanolmetabolic pathway comprises 1J, 1C, 1D, 1E and 1I; and (2) theformaldehyde assimilation pathway comprises 5A and 5B. In oneembodiment, (1) the methanol metabolic pathway comprises 1J, 1C, 1D, 1Fand 1I; and (2) the formaldehyde assimilation pathway comprises 5A and5B. In another embodiment, (1) the methanol metabolic pathway comprises1J, 1L and 1I; and (2) the formaldehyde assimilation pathway comprises5A and 5B. In yet another embodiment, (1) the methanol metabolic pathwaycomprises 1J, 1M, 1N, 1O and 1I; and (2) the formaldehyde assimilationpathway comprises 5A and 5B. In certain embodiments, (1) the methanolmetabolic pathway comprises 1J, 1N, 1O and 1I; and (2) the formaldehydeassimilation pathway comprises 5A and 5B. In some embodiments, (1) themethanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1E and 1I; and (2)the formaldehyde assimilation pathway comprises 5A and 5B. In oneembodiment, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D,1F and 1I; and (2) the formaldehyde assimilation pathway comprises 5Aand 5B. In some embodiments, (1) the methanol metabolic pathwaycomprises 1J, 1C, 1D, 1E and 1G; and (2) the formaldehyde assimilationpathway comprises 5A and 5B. In one embodiment, (1) the methanolmetabolic pathway comprises 1J, 1C, 1D, 1F and 1G; and (2) theformaldehyde assimilation pathway comprises 5A and 5B. In anotherembodiment, (1) the methanol metabolic pathway comprises 1J, 1L and 1G;and (2) the formaldehyde assimilation pathway comprises 5A and 5B. Inyet another embodiment, (1) the methanol metabolic pathway comprises 1J,1M, 1N, 1O and 1G; and (2) the formaldehyde assimilation pathwaycomprises 5A and 5B. In certain embodiments, (1) the methanol metabolicpathway comprises 1J, 1N, 1O and 1G; and (2) the formaldehydeassimilation pathway comprises 5A and 5B. In some embodiments, (1) themethanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1E and 1G; and (2)the formaldehyde assimilation pathway comprises 5A and 5B. In oneembodiment, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D,1F and 1G; and (2) the formaldehyde assimilation pathway comprises 5Aand 5B. In some embodiments, (1) the methanol metabolic pathwaycomprises 1J, 1C, 1D, 1E, 1G and 1H; and (2) the formaldehydeassimilation pathway comprises 5A and 5B. In one embodiment, (1) themethanol metabolic pathway comprises 1J, 1C, 1D, 1F, 1G and 1H; and (2)the formaldehyde assimilation pathway comprises 5A and 5B. In anotherembodiment, (1) the methanol metabolic pathway comprises 1J, 1L, 1G and1H; and (2) the formaldehyde assimilation pathway comprises 5A and 5B.In yet another embodiment, (1) the methanol metabolic pathway comprises1J, 1M, 1N, 1O, 1G and 1H; and (2) the formaldehyde assimilation pathwaycomprises 5A and 5B. In certain embodiments, (1) the methanol metabolicpathway comprises 1J, 1N, 1O, 1G and 1H; and (2) the formaldehydeassimilation pathway comprises 5A and 5B. In some embodiments, (1) themethanol metabolic pathway comprises 1J, 1K, 1C, 1D, 1E, 1G and 1H; and(2) the formaldehyde assimilation pathway comprises 5A and 5B. In oneembodiment, (1) the methanol metabolic pathway comprises 1J, 1K, 1C, 1D,1F, 1G and 1H; and (2) the formaldehyde assimilation pathway comprises5A and 5B. In some embodiments, 1K is spontaneous. In other embodiments,1K is a formaldehyde activating enzyme. In some embodiments, 1M isspontaneous. In some embodiments, the intermediate is a DHA. In otherembodiments, the intermediate is a dihydroxyacetone phosphate. In yetother embodiments, the intermediate is a DHA and a dihydroxyacetonephosphate.

Any methanol metabolic pathway provided herein can be combined with anyformaldehyde assimilation pathway provided herein. In addition, anymethanol metabolic pathway provided herein can be combined with any1,2-propanediol, n-propanol, 1,3-propanediol or glycerol pathway and anyformaldehyde pathway provided herein.

Also provided herein are methods of producing formaldehyde comprisingculturing a non-naturally occurring microbial organism having a methanolmetabolic pathway provided herein. In some embodiments, the methanolmetabolic pathway comprises 1J. In certain embodiments, the organism iscultured in a substantially anaerobic culture medium. In specificembodiments, the formaldehyde is an intermediate that is consumed(assimilated) in the production of 1,2-propanediol, n-propanol,1,3-propanediol, glycerol and other products described herein.

Also provided herein are methods of producing an intermediate ofglycolysis and/or a metabolic pathway that can be used, for example, inthe formation of biomass, comprising culturing a non-naturally occurringmicrobial organism having a methanol metabolic pathway and aformaldehyde assimilation pathway, as provided herein, under conditionsand for a sufficient period of time to produce the intermediate. In someembodiments, the intermediate is a hexylose-6-phosphate. In otherembodiments, the intermediate is a fructose-6-phosphate. In yet otherembodiments, the intermediate is a hexylose-6-phosphate and afructose-6-phosphate. In some embodiments, the intermediate is a DHA. Inother embodiments, the intermediate is a dihydroxyacetone phosphate. Inyet other embodiments, the intermediate is a DHA and a dihydroxyacetonephosphate. In some embodiments, the methanol metabolic pathway comprises1J. In certain embodiments, the organism is cultured in a substantiallyanaerobic culture medium. Such biomass can also be used in methods ofproducing any of the products, such as the biobased products, providedelsewhere herein.

In certain embodiments, the organism comprises two, three, four, five,six or seven exogenous nucleic acids, each encoding a 1,2-propanediol,n-propanol, 1,3-propanediol or glycerol pathway enzyme. In someembodiments, the organism comprises two exogenous nucleic acids, eachencoding a 1,2-propanediol, n-propanol, 1,3-propanediol or glycerolpathway enzyme. In some embodiments, the organism comprises threeexogenous nucleic acids, each encoding a 1,2-propanediol, n-propanol,1,3-propanediol or glycerol pathway enzyme. In some embodiments, theorganism comprises four exogenous nucleic acids, each encoding a1,2-propanediol, n-propanol, 1,3-propanediol or glycerol pathway enzyme.In other embodiments, the organism comprises five exogenous nucleicacids, each encoding a 1,2-propanediol, n-propanol, 1,3-propanediol orglycerol pathway enzyme. In some embodiments, the organism comprises sixexogenous nucleic acids, each encoding a 1,2-propanediol, n-propanol,1,3-propanediol or glycerol pathway enzyme. In other embodiments, theorganism comprises seven exogenous nucleic acids, each encoding a1,2-propanediol, n-propanol, 1,3-propanediol or glycerol pathway enzyme.In certain embodiments, the organism comprises two, three, four, five,six or seven exogenous nucleic acids, each encoding a 1,2-propanediol,n-propanol, 1,3-propanediol or glycerol pathway enzyme; and the organismfurther comprises two, three, four, five, six or seven exogenous nucleicacids, each encoding a methanol metabolic pathway enzyme. In certainembodiments, the organism further comprises two exogenous nucleic acids,each encoding a methanol metabolic pathway enzyme. In certainembodiments, the organism further comprises three exogenous nucleicacids, each encoding a methanol metabolic pathway enzyme. In certainembodiments, the organism comprises further four exogenous nucleicacids, each encoding a methanol metabolic pathway enzyme. In certainembodiments, the organism further comprises five exogenous nucleicacids, each encoding a methanol metabolic pathway enzyme. In certainembodiments, the organism further comprises six exogenous nucleic acids,each encoding a methanol metabolic pathway enzyme. In certainembodiments, the organism further comprises seven exogenous nucleicacids, each encoding a methanol metabolic pathway enzyme.

In some embodiments, the organism comprises two or more exogenousnucleic acids, each encoding a formaldehyde assimilation pathway enzyme.In some embodiments, the organism comprises two exogenous nucleic acids,each encoding a formaldehyde assimilation pathway enzyme. In certainembodiments, the organism comprises two exogenous nucleic acids, eachencoding a formaldehyde assimilation pathway enzyme; and the organismfurther comprises two, three, four, five, six or seven exogenous nucleicacids, each encoding a methanol metabolic pathway enzyme. In certainembodiments, the organism further comprises two exogenous nucleic acids,each encoding a methanol metabolic pathway enzyme. In certainembodiments, the organism further comprises three exogenous nucleicacids, each encoding a methanol metabolic pathway enzyme. In certainembodiments, the organism comprises further four exogenous nucleicacids, each encoding a methanol metabolic pathway enzyme. In certainembodiments, the organism further comprises five exogenous nucleicacids, each encoding a methanol metabolic pathway enzyme. In certainembodiments, the organism further comprises six exogenous nucleic acids,each encoding a methanol metabolic pathway enzyme. In certainembodiments, the organism further comprises seven exogenous nucleicacids, each encoding a methanol metabolic pathway enzyme.

In some embodiments, the at least one exogenous nucleic acid encoding amethanol metabolic pathway enzyme is a heterologous nucleic acid. Inother embodiments, the at least one exogenous nucleic acid encoding aformaldehyde assimilation pathway enzyme is a heterologous nucleic acid.In other embodiments, the at least one exogenous nucleic acid encoding a1,2-propanediol, n-propanol, 1,3-propanediol or glycerol pathway enzymeis a heterologous nucleic acid. In certain embodiments, the at least oneexogenous nucleic acid encoding a methanol metabolic pathway enzyme is aheterologous nucleic acid, and the at least one exogenous nucleic acidencoding a 1,2-propanediol, n-propanol, 1,3-propanediol or glycerolpathway enzyme is a heterologous nucleic acid. In other embodiments, theat least one exogenous nucleic acid encoding a methanol metabolicpathway enzyme is a heterologous nucleic acid, and the at least oneexogenous nucleic acid encoding a formaldehyde assimilation pathwayenzyme is a heterologous nucleic acid.

In certain embodiments, the organism is in a substantially anaerobicculture medium.

It is understood that any of the pathways disclosed herein, as describedin the Examples and exemplified in the figures, including the pathwaysof FIGS. 1, 2, 3, 4 and 5, can be utilized to generate a non-naturallyoccurring microbial organism that produces any pathway intermediate orproduct, as desired. Non-limiting examples of such intermediate orproducts are 1,2-propanediol, n-propanol, 1,3-propanediol or glycerol.As disclosed herein, such a microbial organism that produces anintermediate can be used in combination with another microbial organismexpressing downstream pathway enzymes to produce a desired product.However, it is understood that a non-naturally occurring eukaryoticorganism that produces a 1,2-propanediol, n-propanol, 1,3-propanediol orglycerol pathway intermediate can be utilized to produce theintermediate as a desired product.

In certain embodiments, a non-naturally occurring microbial organismcomprising a methanol metabolic pathway and a 1,2-propanediol,n-propanol, 1,3-propanediol or glycerol pathway provided herein, furthercomprises one or more gene disruptions. In certain embodiments, the oneor more gene disruptions confer increased production of 1,2-propanediol,n-propanol, 1,3-propanediol or glycerol in the organism. In otherembodiments, anon-naturally occurring microbial organism comprising amethanol metabolic pathway and a formaldehyde assimilation pathwayprovided herein, further comprises one or more gene disruptions. In someembodiments, the gene disruption is in an endogenous gene encoding aprotein and/or enzyme involved in native production of ethanol,glycerol, acetate, lactate, formate, CO₂, amino acids, or anycombination thereof, by said microbial organism. In one embodiment, thegene disruption is in an endogenous gene encoding a protein and/orenzyme involved in native production of ethanol. In another embodiment,the gene disruption is in an endogenous gene encoding a protein and/orenzyme involved in native production of glycerol. In other embodiments,the gene disruption is in an endogenous gene encoding a protein and/orenzyme involved in native production of acetate. In another embodiment,the gene disruption is in an endogenous gene encoding a protein and/orenzyme involved in native production of lactate. In one embodiment, thegene disruption is in an endogenous gene encoding a protein and/orenzyme involved in native production of formate. In another embodiment,the gene disruption is in an endogenous gene encoding a protein and/orenzyme involved in native production of CO₂. In other embodiments, thegene disruption is in an endogenous gene encoding a protein and/orenzyme involved in native production of amino acids by said microbialorganism. In some embodiments, the protein or enzyme is a pyruvatedecarboxylase, an ethanol dehydrogenase, a glycerol dehydrogenase, aglycerol-3-phosphatase, a glycerol-3-phosphate dehydrogenase, a lactatedehydrogenase, an acetate kinase, a phosphotransacetylase, a pyruvateoxidase, a pyruvate:quinone oxidoreductase, a pyruvate formate lyase, analcohol dehydrogenase, a lactate dehydrogenase, a pyruvatedehydrogenase, a pyruvate formate-lyase-2-ketobutyrate formate-lyase, apyruvate transporter, a monocarboxylate transporter, a NADHdehydrogenase, a cytochrome oxidase, a pyruvate kinase, or anycombination thereof. In certain embodiments, the organism comprises fromone to twenty-five gene disruptions. In other embodiments, the organismcomprises from one to twenty gene disruptions. In some embodiments, theorganism comprises from one to fifteen gene disruptions. In otherembodiments, the organism comprises from one to ten gene disruptions. Insome embodiments, the organism comprises from one to five genedisruptions. In certain embodiments, the organism comprises 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24 or 25 gene disruptions or more.

In other embodiments, a non-naturally occurring microbial organismcomprising a methanol metabolic pathway and a 1,2-propanediol,n-propanol, 1,3-propanediol or glycerol pathway provided herein, furthercomprises one or more endogenous proteins or enzymes involved in nativeproduction of ethanol, glycerol, acetate, lactate, formate, CO₂ and/oramino acids by said microbial organism, wherein said one or moreendogenous proteins or enzymes has attenuated protein or enzyme activityand/or expression levels. In some embodiments, a non-naturally occurringmicrobial organism comprising a methanol metabolic pathway and aformaldehyde assimilation pathway provided herein, further comprises oneor more endogenous proteins or enzymes involved in native production ofethanol, glycerol, acetate, lactate, formate, CO2 and/or amino acids bysaid microbial organism, wherein said one or more endogenous proteins orenzymes has attenuated protein or enzyme activity and/or expressionlevels. In one embodiment the endogenous protein or enzyme is a pyruvatedecarboxylase, an ethanol dehydrogenase, a glycerol dehydrogenase, aglycerol-3-phosphatase, a glycerol-3-phosphate dehydrogenase, a lactatedehydrogenase, an acetate kinase, a phosphotransacetylase, a pyruvateoxidase, a pyruvate:quinone oxidoreductase, a pyruvate formate lyase, analcohol dehydrogenase, a lactate dehydrogenase, a pyruvatedehydrogenase, a pyruvate formate-lyase-2-ketobutyrate formate-lyase, apyruvate transporter, a monocarboxylate transporter, a NADHdehydrogenase, a cytochrome oxidase, a pyruvate kinase, or anycombination thereof.

Each of the non-naturally occurring alterations provided herein resultin increased production and an enhanced level of 1,2-propanediol,n-propanol, 1,3-propanediol or glycerol, for example, during theexponential growth phase of the microbial organism, compared to a strainthat does not contain such metabolic alterations, under appropriateculture conditions. Appropriate conditions include, for example, thosedisclosed herein, including conditions such as particular carbon sourcesor reactant availabilities and/or adaptive evolution.

Given the teachings and guidance provided herein, those skilled in theart will understand that to introduce a metabolic alteration, such asattenuation of an enzyme, it can be necessary to disrupt the catalyticactivity of the one or more enzymes involved in the reaction.Alternatively, a metabolic alteration can include disrupting expressionof a regulatory protein or cofactor necessary for enzyme activity ormaximal activity. Furthermore, genetic loss of a cofactor necessary foran enzymatic reaction can also have the same effect as a disruption ofthe gene encoding the enzyme. Disruption can occur by a variety ofmethods including, for example, deletion of an encoding gene orincorporation of a genetic alteration in one or more of the encodinggene sequences. The encoding genes targeted for disruption can be one,some, or all of the genes encoding enzymes involved in the catalyticactivity. For example, where a single enzyme is involved in a targetedcatalytic activity, disruption can occur by a genetic alteration thatreduces or eliminates the catalytic activity of the encoded geneproduct. Similarly, where the single enzyme is multimeric, includingheteromeric, disruption can occur by a genetic alteration that reducesor destroys the function of one or all subunits of the encoded geneproducts. Destruction of activity can be accomplished by loss of thebinding activity of one or more subunits required to form an activecomplex, by destruction of the catalytic subunit of the multimericcomplex or by both. Other functions of multimeric protein associationand activity also can be targeted in order to disrupt a metabolicreaction of the invention. Such other functions are well known to thoseskilled in the art. Similarly, a target enzyme activity can be reducedor eliminated by disrupting expression of a protein or enzyme thatmodifies and/or activates the target enzyme, for example, a moleculerequired to convert an apoenzyme to a holoenzyme. Further, some or allof the functions of a single polypeptide or multimeric complex can bedisrupted according to the invention in order to reduce or abolish thecatalytic activity of one or more enzymes involved in a reaction ormetabolic modification of the invention. Similarly, some or all ofenzymes involved in a reaction or metabolic modification of theinvention can be disrupted so long as the targeted reaction is reducedor eliminated.

Given the teachings and guidance provided herein, those skilled in theart also will understand that an enzymatic reaction can be disrupted byreducing or eliminating reactions encoded by a common gene and/or by oneor more orthologs of that gene exhibiting similar or substantially thesame activity. Reduction of both the common gene and all orthologs canlead to complete abolishment of any catalytic activity of a targetedreaction. However, disruption of either the common gene or one or moreorthologs can lead to a reduction in the catalytic activity of thetargeted reaction sufficient to promote coupling of growth to productbiosynthesis. Exemplified herein are both the common genes encodingcatalytic activities for a variety of metabolic modifications as well astheir orthologs. Those skilled in the art will understand thatdisruption of some or all of the genes encoding a enzyme of a targetedmetabolic reaction can be practiced in the methods of the invention andincorporated into the non-naturally occurring microbial organisms of theinvention in order to achieve the increased production of fatty alcohol,fatty aldehyde or fatty acid or growth-coupled product production.

Given the teachings and guidance provided herein, those skilled in theart also will understand that enzymatic activity or expression can beattenuated using well known methods. Reduction of the activity or amountof an enzyme can mimic complete disruption if the reduction causesactivity of the enzyme to fall below a critical level that is normallyrequired for the pathway to function. Reduction of enzymatic activity byvarious techniques rather than disruption can be important for anorganism's viability. Methods of reducing enzymatic activity that resultin similar or identical effects of a gene disruption include, but arenot limited to: reducing gene transcription or translation;destabilizing mRNA, protein or catalytic RNA; and mutating a gene thataffects enzyme kinetics. Natural or imposed regulatory controls can alsoaccomplish enzyme attenuation including: promoter replacement; loss oralteration of transcription factors; introduction of inhibitory RNAs orpeptides such as siRNA, antisense RNA, RNA or peptide/small-moleculebinding aptamers, ribozymes, aptazymes and riboswitches; and addition ofdrugs and other chemicals that reduce or disrupt enzymatic activity suchas gene splicing.

One of ordinary skill in the art will also recognize that attenuation ofan enzyme can be done at various levels. For example, at the gene level,mutations causing a partial or complete null phenotype or epistaticgenetic effects that mask the activity of a gene product can be used toattenuate an enzyme. At the gene expression level, methods forattenuation include: coupling transcription to an endogenous orexogenous inducer such as IPTG, then adding low or 0 levels of inducerduring the production phase; introducing or modifying positive ornegative regulators; modify histone acetylation/deacetylation in regionwhere gene is integrated; introducing a transposition to disrupt apromoter or a regulatory gene; flipping of a transposable element orpromoter region; deleting one allele resulting in loss of heterozygosityin a diploid organism; introducing nucleic acids that increase RNAdegradation; or in bacteria, for example, introduction of a tmRNA tag,which can lead to RNA degradation and ribosomal stalling. At thetranslational level, attenuation can include: introducing rare codons tolimit translation; introducing RNA interference molecules that blocktranslation; modifying regions outside the coding sequence, such asintroducing secondary structure into UTR regions to block translation orreduce efficiency of translation; adding RNAase sites for rapidtranscript degradation; introducing antisense RNA oligomers or antisensetranscripts; introducing RNA or peptide aptamers, ribozymes, aptazymes,riboswitches; or introducing translational regulatory elements involvingRNA structure that can prevent or reduce translation that can becontrolled by the presence or absence of small molecules. At the levelof enzyme localization and/or longevity, enzyme attenuation can include:adding a degradation tag for faster protein turnover; or adding alocalization tag that results in the enzyme being localized to acompartment where it would not be able to react normally. At the levelof post-translational regulation, enzyme attenuation can include:increasing intracellular concentration of known inhibitors; or modifyingpost-translational modified sites. At the level of enzyme activity,enzyme attenuation can include: adding endogenous or exogenousinhibitor, such as a target-specific drug, to reduce enzyme activity;limiting availability of essential cofactors, such as B12, for an enzymethat require it; chelating a metal ion that is required for activity; orintroducing a dominant negative mutation.

In some embodiments, microaerobic designs can be used based on thegrowth-coupled formation of the desired product. To examine this,production cones can be constructed for each strategy by firstmaximizing and, subsequently minimizing the product yields at differentrates of biomass formation feasible in the network. If the rightmostboundary of all possible phenotypes of the mutant network is a singlepoint, it implies that there is a unique optimum yield of the product atthe maximum biomass formation rate possible in the network. In othercases, the rightmost boundary of the feasible phenotypes is a verticalline, indicating that at the point of maximum biomass the network canmake any amount of the product in the calculated range, including thelowest amount at the bottommost point of the vertical line. Such designsare given a low priority.

The 1,2-propanediol-, n-propanol-, 1,3-propanediol- orglycerol-production strategies provided herein can be disrupted toincrease production of 1,2-propanediol, n-propanol, 1,3-propanediol orglycerol. Accordingly, also provided herein is a non-naturally occurringmicrobial organism having metabolic modifications coupling1,2-propanediol, n-propanol, 1,3-propanediol or glycerol production togrowth of the organism, where the metabolic modifications includesdisruption of one or more genes selected from the genes encodingproteins and/or enzymes provided herein.

Each of the strains can be supplemented with additional deletions if itis determined that the strain designs do not sufficiently increase theproduction of 1,2-propanediol, n-propanol, 1,3-propanediol or glyceroland/or couple the formation of the product with biomass formation.Alternatively, some other enzymes not known to possess significantactivity under the growth conditions can become active due to adaptiveevolution or random mutagenesis. Such activities can also be knockedout. However, gene deletions provided herein allow the construction ofstrains exhibiting high-yield production of 1,2-propanediol, n-propanol,1,3-propanediol or glycerol, including growth-coupled production of1,2-propanediol, n-propanol, 1,3-propanediol or glycerol.

In another aspect, provided herein is a method for producing1,2-propanediol, n-propanol, 1,3-propanediol or glycerol, comprisingculturing any one of the non-naturally occurring microbial organismscomprising a methanol metabolic pathway and a 1,2-propanediol,n-propanol, 1,3-propanediol or glycerol pathway provided herein underconditions and for a sufficient period of time to produce1,2-propanediol, n-propanol, 1,3-propanediol or glycerol. In certainembodiments, the organism is cultured in a substantially anaerobicculture medium.

In one embodiment, provided herein are methods for producing1,2-propanediol, comprising culturing an organism provided herein (e.g.,a non-naturally occurring microbial organisms comprising a methanolmetabolic pathway and a 1,2-propanediol pathway) under conditions andfor a sufficient period of time to produce 1,2-propanediol. In someembodiments, the method comprises culturing, for a sufficient period oftime to produce 1,2-propanediol, a non-naturally occurring microbialorganism, comprising (1) a methanol metabolic pathway, wherein saidorganism comprises at least one exogenous nucleic acid encoding amethanol metabolic pathway enzyme in a sufficient amount to enhance theavailability of reducing equivalents in the presence of methanol; and(2) a 1,2-propanediol pathway, comprising at least one exogenous nucleicacid encoding a 1,2-propanediol pathway enzyme expressed in a sufficientamount to produce 1,2-propanediol.

In another embodiment, provided herein are methods for producingn-propanol, comprising culturing an organism provided herein (e.g., anon-naturally occurring microbial organisms comprising a methanolmetabolic pathway and an n-propanol pathway) under conditions and for asufficient period of time to produce n-propanol. In some embodiments,the method comprises culturing, for a sufficient period of time toproduce n-propanol, a non-naturally occurring microbial organism,comprising (1) a methanol metabolic pathway, wherein said organismcomprises at least one exogenous nucleic acid encoding a methanolmetabolic pathway enzyme in a sufficient amount to enhance theavailability of reducing equivalents in the presence of methanol; and(2) an n-propanol pathway, comprising at least one exogenous nucleicacid encoding an n-propanol pathway enzyme expressed in a sufficientamount to produce n-propanol.

In other embodiments, provided herein are methods for producing1,3-propanediol, comprising culturing an organism provided herein (e.g.,a non-naturally occurring microbial organisms comprising a methanolmetabolic pathway and an 1,3-propanediol pathway) under conditions andfor a sufficient period of time to produce 1,3-propanediol. In someembodiments, the method comprises culturing, for a sufficient period oftime to produce 1,3-propanediol, a non-naturally occurring microbialorganism, comprising (1) a methanol metabolic pathway, wherein saidorganism comprises at least one exogenous nucleic acid encoding amethanol metabolic pathway enzyme in a sufficient amount to enhance theavailability of reducing equivalents in the presence of methanol; and(2) an 1,3-propanediol pathway, comprising at least one exogenousnucleic acid encoding an 1,3-propanediol pathway enzyme expressed in asufficient amount to produce 1,3-propanediol.

In yet other embodiments, provided herein are methods for producingglycerol, comprising culturing an organism provided herein (e.g., anon-naturally occurring microbial organisms comprising a methanolmetabolic pathway and an glycerol pathway) under conditions and for asufficient period of time to produce glycerol. In some embodiments, themethod comprises culturing, for a sufficient period of time to produceglycerol, a non-naturally occurring microbial organism, comprising (1) amethanol metabolic pathway, wherein said organism comprises at least oneexogenous nucleic acid encoding a methanol metabolic pathway enzyme in asufficient amount to enhance the availability of reducing equivalents inthe presence of methanol; and (2) an glycerol pathway, comprising atleast one exogenous nucleic acid encoding an glycerol pathway enzymeexpressed in a sufficient amount to produce glycerol.

In certain embodiments of the methods provided herein, the organismfurther comprises at least one nucleic acid encoding a 1,2-propanediol,n-propanol, 1,3-propanediol or glycerol pathway enzyme expressed in asufficient amount to produce 1,2-propanediol, n-propanol,1,3-propanediol or glycerol. In some embodiments, the nucleic acid is anexogenous nucleic acid, in other embodiments. the nucleic acid is anendogenous nucleic acid. In some embodiments, the organism furthercomprises one or more gene disruptions provided herein that conferincreased production of 1,2-propanediol, n-propanol, 1,3-propanediol orglycerol in the organism. In certain embodiments, the one or more genedisruptions occurs in an endogenous gene encoding a protein or enzymeinvolved in native production of ethanol, glycerol, acetate, lactate,formate, CO₂ and/or amino acids by said microbial organism. In otherembodiments, the organism further comprises one or more endogenousproteins or enzymes involved in native production of ethanol, glycerol,acetate, lactate, formate, CO₂ and/or amino acids by said microbialorganism, wherein said one or more endogenous proteins or enzymes hasattenuated protein or enzyme activity and/or expression levels. Incertain embodiments, the organism is a Crabtree positive, eukaryoticorganism, and the organism is cultured in a culture medium comprisingglucose. In certain embodiments, the organism comprises from one totwenty-five gene disruptions. In other embodiments, the organismcomprises from one to twenty gene disruptions. In some embodiments, theorganism comprises from one to fifteen gene disruptions. In otherembodiments, the organism comprises from one to ten gene disruptions. Insome embodiments, the organism comprises from one to five genedisruptions. In certain embodiments, the organism comprises 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24 or 25 gene disruptions or more.

In an additional embodiment, the invention provides a non-naturallyoccurring microbial organism having a 1,2-propanediol, n-propanol,1,3-propanediol or glycerol pathway, formaldehyde assimilation pathwayand/or methanol metabolic pathway, wherein the non-naturally occurringmicrobial organism comprises at least one exogenous nucleic acidencoding an enzyme or protein that converts a substrate to a product. Byway of example, in FIG. 1, the substrate of 1J is methanol, and theproduct is formaldehyde; the substrate of 1L is formaldehyde, and theproduct is formate; and so forth. One skilled in the art will understandthat these are merely exemplary and that any of the substrate-productpairs disclosed herein suitable to produce a desired product and forwhich an appropriate activity is available for the conversion of thesubstrate to the product can be readily determined by one skilled in theart based on the teachings herein. Thus, provided herein arenon-naturally occurring microbial organisms containing at least oneexogenous nucleic acid encoding an enzyme or protein, where the enzymeor protein converts the substrates and products of a methanol metabolicpathway, such as that shown in FIG. 1; a, 2-propanediol, n-propanol,1,3-propanediol or glycerol pathway, such as that shown in FIG. 2 or 3;and/or a formaldehyde assimilation pathway, such as that shown in FIG. 4or 5.

While generally described herein as a microbial organism that contains a1,2-propanediol, n-propanol, 1,3-propanediol or glycerol pathway,formaldehyde assimilation pathway, and/or a methanol metabolic pathway,it is understood that provided herein are also non-naturally occurringmicrobial organism comprising at least one exogenous nucleic acidencoding a 1,2-propanediol, n-propanol, 1,3-propanediol or glycerolpathway, formaldehyde assimilation pathway, and/or a methanol metabolicpathway enzyme expressed in a sufficient amount to produce anintermediate of a 1,2-propanediol, n-propanol, 1,3-propanediol orglycerol, pathway, formaldehyde assimilation pathway, and/or a methanolmetabolic pathway intermediate. For example, as disclosed herein, a1,2-propanediol, n-propanol, 1,3-propanediol or glycerol pathway isexemplified in FIGS. 2 and 3. Therefore, in addition to a microbialorganism containing a 1,2-propanediol, n-propanol, 1,3-propanediol orglycerol pathway that produces 1,2-propanediol, n-propanol,1,3-propanediol or glycerol, also provided herein is a non-naturallyoccurring microbial organism comprising at least one exogenous nucleicacid encoding a 1,2-propanediol, n-propanol, 1,3-propanediol or glycerolpathway enzyme, where the microbial organism produces a 1,2-propanediol,n-propanol, 1,3-propanediol or glycerol pathway intermediate.

In some embodiments, the carbon feedstock and other cellular uptakesources such as phosphate, ammonia, sulfate, chloride and other halogenscan be chosen to alter the isotopic distribution of the atoms present in1,2-propanediol, n-propanol, 1,3-propanediol and/or glycerol or any1,2-propanediol, n-propanol, 1,3-propanediol and/or glycerol pathwayintermediate. The various carbon feedstock and other uptake sourcesenumerated above will be referred to herein, collectively, as “uptakesources.” Uptake sources can provide isotopic enrichment for any atompresent in the product 1,2-propanediol, n-propanol, 1,3-propanediol or aglycerol and/or 1,2-propanediol, n-propanol, 1,3-propanediol or glycerolpathway intermediate, or for side products generated in reactionsdiverging away from a 1,2-propanediol, n-propanol, 1,3-propanedioland/or glycerol pathway. Isotopic enrichment can be achieved for anytarget atom including, for example, carbon, hydrogen, oxygen, nitrogen,sulfur, phosphorus, chloride or other halogens.

In some embodiments, the uptake sources can be selected to alter thecarbon-12, carbon-13, and carbon-14 ratios. In some embodiments, theuptake sources can be selected to alter the oxygen-16, oxygen-17, andoxygen-18 ratios. In some embodiments, the uptake sources can beselected to alter the hydrogen, deuterium, and tritium ratios. In someembodiments, the uptake sources can selected to alter the nitrogen-14and nitrogen-15 ratios. In some embodiments, the uptake sources can beselected to alter the sulfur-32, sulfur-33, sulfur-34, and sulfur-35ratios. In some embodiments, the uptake sources can be selected to alterthe phosphorus-31, phosphorus-32, and phosphorus-33 ratios. In someembodiments, the uptake sources can be selected to alter thechlorine-35, chlorine-36, and chlorine-37 ratios.

In some embodiments, the isotopic ratio of a target atom can be variedto a desired ratio by selecting one or more uptake sources. An uptakesource can be derived from a natural source, as found in nature, or froma man-made source, and one skilled in the art can select a naturalsource, a man-made source, or a combination thereof, to achieve adesired isotopic ratio of a target atom. An example of a man-made uptakesource includes, for example, an uptake source that is at leastpartially derived from a chemical synthetic reaction. Such isotopicallyenriched uptake sources can be purchased commercially or prepared in thelaboratory and/or optionally mixed with a natural source of the uptakesource to achieve a desired isotopic ratio. In some embodiments, atarget isotopic ratio of an uptake source can be obtained by selecting adesired origin of the uptake source as found in nature For example, asdiscussed herein, a natural source can be a biobased derived from orsynthesized by a biological organism or a source such as petroleum-basedproducts or the atmosphere. In some such embodiments, a source ofcarbon, for example, can be selected from a fossil fuel-derived carbonsource, which can be relatively depleted of carbon-14, or anenvironmental or atmospheric carbon source, such as CO₂, which canpossess a larger amount of carbon-14 than its petroleum-derivedcounterpart.

Isotopic enrichment is readily assessed by mass spectrometry usingtechniques known in the art such as Stable Isotope Ratio MassSpectrometry (SIRMS) and Site-Specific Natural Isotopic Fractionation byNuclear Magnetic Resonance (SNIF-NMR). Such mass spectral techniques canbe integrated with separation techniques such as liquid chromatography(LC) and/or high performance liquid chromatography (HPLC).

The unstable carbon isotope carbon-14 or radiocarbon makes up forroughly 1 in 10¹² carbon atoms in the earth's atmosphere and has ahalf-life of about 5700 years. The stock of carbon is replenished in theupper atmosphere by a nuclear reaction involving cosmic rays andordinary nitrogen (¹⁴N). Fossil fuels contain no carbon-14, as itdecayed long ago. Burning of fossil fuels lowers the atmosphericcarbon-14 fraction, the so-called “Suess effect”.

Methods of determining the isotopic ratios of atoms in a compound arewell known to those skilled in the art. Isotopic enrichment is readilyassessed by mass spectrometry using techniques known in the art such asaccelerated mass spectrometry (AMS), Stable Isotope Ratio MassSpectrometry (SIRMS) and Site-Specific Natural Isotopic Fractionation byNuclear Magnetic Resonance (SNIF-NMR). Such mass spectral techniques canbe integrated with separation techniques such as liquid chromatography(LC), high performance liquid chromatography (HPLC) and/or gaschromatography, and the like.

In the case of carbon, ASTM D6866 was developed in the United States asa standardized analytical method for determining the biobased content ofsolid, liquid, and gaseous samples using radiocarbon dating by theAmerican Society for Testing and Materials (ASTM) International. Thestandard is based on the use of radiocarbon dating for the determinationof a product's biobased content. ASTM D6866 was first published in 2004,and the current active version of the standard is ASTM D6866-11(effective Apr. 1, 2011). Radiocarbon dating techniques are well knownto those skilled in the art, including those described herein.

The biobased content of a compound is estimated by the ratio ofcarbon-14 (¹⁴C) to carbon-12 (¹²C). Specifically, the Fraction Modern(Fm) is computed from the expression: Fm=(S−B)/(M−B), where B, S and Mrepresent the ¹⁴C/¹²C ratios of the blank, the sample and the modernreference, respectively. Fraction Modern is a measurement of thedeviation of the ¹⁴C/¹²C ratio of a sample from “Modern.” Modern isdefined as 95% of the radiocarbon concentration (in AD 1950) of NationalBureau of Standards (NBS) Oxalic Acid I (i.e., standard referencematerials (SRM) 4990b) normalized to δ¹³C_(VPDB)=−19 per mil (Olsson,The use of Oxalic acid as a Standard. in, Radiocarbon Variations andAbsolute Chronology, Nobel Symposium. 12th Proc., John Wiley & Sons, NewYork (1970)). Mass spectrometry results, for example, measured by ASM,are calculated using the internationally agreed upon definition of 0.95times the specific activity of NBS Oxalic Acid I (SRM 4990b) normalizedto δ¹³C_(VPDB)=−19 per mil. This is equivalent to an absolute (AD 1950)¹⁴C/¹²C ratio of 1.176±0.010×10⁻¹² (Karlen et al., Arkiv Geofysik,4:465-471 (1968)). The standard calculations take into account thedifferential uptake of one isotope with respect to another, for example,the preferential uptake in biological systems of C¹² over C¹³ over C¹⁴,and these corrections are reflected as a Fm corrected for δ¹³.

An oxalic acid standard (SRM 4990b or HOx 1) was made from a crop of1955 sugar beet. Although there were 1000 lbs made, this oxalic acidstandard is no longer commercially available. The Oxalic Acid IIstandard (HOx 2; N.I.S.T designation SRM 4990 C) was made from a crop of1977 French beet molasses. In the early 1980's, a group of 12laboratories measured the ratios of the two standards. The ratio of theactivity of Oxalic acid II to 1 is 1.2933±0.001 (the weighted mean). Theisotopic ratio of HOx II is −17.8 per mille. ASTM D6866-11 suggests useof the available Oxalic Acid II standard SRM 4990 C (Hox2) for themodern standard (see discussion of original vs. currently availableoxalic acid standards in Mann, Radiocarbon, 25(2):519-527 (1983)). AFm=0% represents the entire lack of carbon-14 atoms in a material, thusindicating a fossil (for example, petroleum based) carbon source. AFm=100%, after correction for the post-1950 injection of carbon-14 intothe atmosphere from nuclear bomb testing, indicates an entirely moderncarbon source. As described herein, such a “modern” source includesbiobased sources.

As described in ASTM D6866, the percent modern carbon (pMC) can begreater than 100% because of the continuing but diminishing effects ofthe 1950s nuclear testing programs, which resulted in a considerableenrichment of carbon-14 in the atmosphere as described in ASTM D6866-11.Because all sample carbon-14 activities are referenced to a “pre-bomb”standard, and because nearly all new biobased products are produced in apost-bomb environment, all pMC values (after correction for isotopicfraction) must be multiplied by 0.95 (as of 2010) to better reflect thetrue biobased content of the sample. A biobased content that is greaterthan 103% suggests that either an analytical error has occurred, or thatthe source of biobased carbon is more than several years old.

ASTM D6866 quantities the biobased content relative to the material'stotal organic content and does not consider the inorganic carbon andother non-carbon containing substances present. For example, a productthat is 50% starch-based material and 50% water would be considered tohave a Biobased Content=100% (50% organic content that is 100% biobased)based on ASTM D6866. In another example, a product that is 50%starch-based material, 25% petroleum-based, and 25% water would have aBiobased Content=66.7% (75% organic content but only 50% of the productis biobased). In another example, a product that is 50% organic carbonand is a petroleum-based product would be considered to have a BiobasedContent=0% (50% organic carbon but from fossil sources). Thus, based onthe well known methods and known standards for determining the biobasedcontent of a compound or material, one skilled in the art can readilydetermine the biobased content and/or prepared downstream products thatutilize of the invention having a desired biobased content.

Applications of carbon-14 dating techniques to quantify bio-basedcontent of materials are known in the art (Currie et al., NuclearInstruments and Methods in Physics Research B, 172:281-287 (2000)). Forexample, carbon-14 dating has been used to quantify bio-based content interephthalate-containing materials (Colonna et al., Green Chemistry,13:2543-2548 (2011)). Notably, polypropylene terephthalate (PPT)polymers derived from renewable 1,3-propanediol and petroleum-derivedterephthalic acid resulted in Fm values near 30% (i.e., since 3/11 ofthe polymeric carbon derives from renewable 1,3-propanediol and 8/11from the fossil end member terephthalic acid) (Currie et al., supra,2000). In contrast, polybutylene terephthalate polymer derived from bothrenewable BDO and renewable terephthalic acid resulted in bio-basedcontent exceeding 90% (Colonna et al., supra, 2011).

Accordingly, in some embodiments, the present invention provides1,2-propanediol, n-propanol, 1,3-propanediol or glycerol, or a1,2-propanediol, n-propanol, 1,3-propanediol or glycerol pathwayintermediate thereof, that has a carbon-12, carbon-13, and carbon-14ratio that reflects an atmospheric carbon, also referred to asenvironmental carbon, uptake source. For example, in some aspects, the1,2-propanediol, n-propanol, 1,3-propanediol or glycerol, or a1,2-propanediol, n-propanol, 1,3-propanediol or glycerol intermediatethereof can have an Fm value of at least 10%, at least 15%, at least20%, at least 25%, at least 30%, at least 35%, at least 40%, at least45%, at least 50%, at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 98% or as much as 100%. In some such embodiments, theuptake source is CO₂. In some embodiments, the present inventionprovides 1,2-propanediol, n-propanol, 1,3-propanediol or glycerol, or a1,2-propanediol, n-propanol, 1,3-propanediol or glycerol intermediatethereof, that has a carbon-12, carbon-13, and carbon-14 ratio thatreflects petroleum-based carbon uptake source. In this aspect, the a1,2-propanediol, n-propanol, 1,3-propanediol or glycerol, or a1,2-propanediol, n-propanol, 1,3-propanediol or glycerol intermediatecan have an Fm value of less than 95%, less than 90%, less than 85%,less than 80%, less than 75%, less than 70%, less than 65%, less than60%, less than 55%, less than 50%, less than 45%, less than 40%, lessthan 35%, less than 30%, less than 25%, less than 20%, less than 15%,less than 10%, less than 5%, less than 2% or less than 1%. In someembodiments, the present invention provides a 1,2-propanediol,n-propanol, 1,3-propanediol or glycerol, or a 1,2-propanediol,n-propanol, 1,3-propanediol or glycerol intermediate thereof, that has acarbon-12, carbon-13, and carbon-14 ratio that is obtained by acombination of an atmospheric carbon uptake source with apetroleum-based uptake source. Using such a combination of uptakesources is one way by which the carbon-12, carbon-13, and carbon-14ratio can be varied, and the respective ratios would reflect theproportions of the uptake sources.

Further, the present invention relates to biologically produced1,2-propanediol, n-propanol, 1,3-propanediol or glycerol, or a1,2-propanediol, n-propanol, 1,3-propanediol or glycerol intermediatethereof, as disclosed herein, and to the products derived therefrom,wherein the a 1,2-propanediol, n-propanol, 1,3-propanediol or glycerol,or an intermediate thereof, has a carbon-12, carbon-13, and carbon-14isotope ratio of about the same value as the CO₂ that occurs in theenvironment. For example, in some aspects the invention providesbioderived 1,2-propanediol, n-propanol, 1,3-propanediol or glycerol, oran intermediate thereof, having a carbon-12 versus carbon-13 versuscarbon-14 isotope ratio of about the same value as the CO₂ that occursin the environment, or any of the other ratios disclosed herein. It isunderstood, as disclosed herein, that a product can have a carbon-12versus carbon-13 versus carbon-14 isotope ratio of about the same valueas the CO₂ that occurs in the environment, or any of the ratiosdisclosed herein, wherein the product is generated from bioderived1,2-propanediol, n-propanol, 1,3-propanediol or glycerol, or anintermediate thereof, as disclosed herein, wherein the bioderivedproduct is chemically modified to generate a final product. Methods ofchemically modifying a bioderived product of 1,2-propanediol,n-propanol, 1,3-propanediol or glycerol, or an intermediate thereof, togenerate a desired product are well known to those skilled in the art,as described herein. The invention further provides pharmaceuticalformulations, humectants, solvents, sweeteners, preservative, foodadditives, monoglycerides, diglycerides, emulsifiers, antifreeze andde-icer agents, oil dispersants, solvents, resins, polyglycerol esters,moisturizer, oils, shortenings, margarines, and medical, personal care,cosmetic or pharmaceutical preparations, and the like, having acarbon-12 versus carbon-13 versus carbon-14 isotope ratio of about thesame value as the CO₂ that occurs in the environment, wherein thepharmaceutical formulations, humectants, solvents, sweeteners,preservative, food additives, monoglycerides, diglycerides, emulsifiers,antifreeze and de-icer agents, oil dispersants, solvents, resins,polyglycerol esters, moisturizer, oils, shortenings, margarines, andmedical, personal care, cosmetic or pharmaceutical preparations, and thelike, are generated directly from or in combination with bioderived1,2-propanediol, n-propanol, 1,3-propanediol or glycerol or a bioderivedintermediate thereof, as disclosed herein.

1,2-propanediol, n-propanol, 1,3-propanediol and glycerol, as well asintermediates thereof, are chemicals used in commercial and industrialapplications. Non-limiting examples of such applications includeproduction of pharmaceutical formulations, humectants, solvents,sweeteners, preservative, food additives, monoglycerides, diglycerides,emulsifiers, antifreeze and de-icer agents, oil dispersants, solvents,resins, polyglycerol esters, moisturizer, oils, shortenings, margarines,and medical, personal care, cosmetic or pharmaceutical preparations, andthe like. Moreover, 1,2-propanediol, n-propanol, 1,3-propanediol andglycerol are also used as a raw material in the production of a widerange of products including pharmaceutical formulations, humectants,solvents, sweeteners, preservative, food additives, monoglycerides,diglycerides, emulsifiers, antifreeze and de-icer agents, oildispersants, solvents, resins, polyglycerol esters, moisturizer, oils,shortenings, margarines, and medical, personal care, cosmetic orpharmaceutical preparations, and the like. Accordingly, in someembodiments, the invention provides biobased pharmaceuticalformulations, humectants, solvents, sweeteners, preservative, foodadditives, monoglycerides, diglycerides, emulsifiers, antifreeze andde-icer agents, oil dispersants, solvents, resins, polyglycerol esters,moisturizer, oils, shortenings, margarines, and medical, personal care,cosmetic or pharmaceutical preparations, and the like, comprising one ormore of bioderived 1,2-propanediol, n-propanol, 1,3-propanediol orglycerol, or a bioderived intermediate thereof, produced by anon-naturally occurring microorganism of the invention or produced usinga method disclosed herein.

As used herein, the term “bioderived” means derived from or synthesizedby a biological organism and can be considered a renewable resourcesince it can be generated by a biological organism. Such a biologicalorganism, in particular the microbial organisms of the inventiondisclosed herein, can utilize feedstock or biomass, such as, sugars orcarbohydrates obtained from an agricultural, plant, bacterial, or animalsource. Alternatively, the biological organism can utilize atmosphericcarbon. As used herein, the term “biobased” means a product as describedabove that is composed, in whole or in part, of a bioderived compound ofthe invention. A biobased or bioderived product is in contrast to apetroleum derived product, wherein such a product is derived from orsynthesized from petroleum or a petrochemical feedstock.

In some embodiments, the invention provides pharmaceutical formulations,humectants, solvents, sweeteners, preservative, food additives,monoglycerides, diglycerides, emulsifiers, antifreeze and de-iceragents, oil dispersants, solvents, resins, polyglycerol esters,moisturizer, oils, shortenings, margarines, and medical, personal care,cosmetic or pharmaceutical preparations, and the like, comprisingbioderived 1,2-propanediol, n-propanol, 1,3-propanediol or glycerol, ora bioderived intermediate thereof, wherein the bioderived1,2-propanediol, n-propanol, 1,3-propanediol or glycerol, or bioderivedintermediate thereof, includes all or part of the a 1,2-propanediol,n-propanol, 1,3-propanediol or glycerol, or an intermediate thereof,used in the production of pharmaceutical formulations, humectants,solvents, sweeteners, preservative, food additives, monoglycerides,diglycerides, emulsifiers, antifreeze and de-icer agents, oildispersants, solvents, resins, polyglycerol esters, moisturizer, oils,shortenings, margarines, and medical, personal care, cosmetic orpharmaceutical preparations, and the like. Thus, in some aspects, theinvention provides a biobased pharmaceutical formulations, humectants,solvents, sweeteners, preservative, food additives, monoglycerides,diglycerides, emulsifiers, antifreeze and de-icer agents, oildispersants, solvents, resins, polyglycerol esters, moisturizer, oils,shortenings, margarines, and medical, personal care, cosmetic orpharmaceutical preparations, and the like, comprising at least 2%, atleast 3%, at least 5%, at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, at least 35%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 95%, atleast 98% or 100% bioderived 1,2-propanediol, n-propanol,1,3-propanediol or glycerol, or a bioderived 1,2-propanediol,n-propanol, 1,3-propanediol or glycerol intermediate, as disclosedherein. Additionally, in some aspects, the invention provides biobasedpharmaceutical formulations, humectants, solvents, sweeteners,preservative, food additives, monoglycerides, diglycerides, emulsifiers,antifreeze and de-icer agents, oil dispersants, solvents, resins,polyglycerol esters, moisturizer, oils, shortenings, margarines, andmedical, personal care, cosmetic or pharmaceutical preparations, and thelike, wherein the a 1,2-propanediol, n-propanol, 1,3-propanediol orglycerol, or a 1,2-propanediol, n-propanol, 1,3-propanediol or glycerolintermediate, used in its production is a combination of bioderived andpetroleum derived 1,2-propanediol, n-propanol, 1,3-propanediol orglycerol, or a 1,2-propanediol, n-propanol, 1,3-propanediol or glycerolintermediate thereof. For example, biobased pharmaceutical formulations,humectants, solvents, sweeteners, preservative, food additives,monoglycerides, diglycerides, emulsifiers, antifreeze and de-iceragents, oil dispersants, solvents, resins, polyglycerol esters,moisturizer, oils, shortenings, margarines, and medical, personal care,cosmetic or pharmaceutical preparations, and the like, can be producedusing 50% bioderived 1,2-propanediol, n-propanol, 1,3-propanediol orglycerol and 50% petroleum derived 1,2-propanediol, n-propanol,1,3-propanediol or glycerol or other desired ratios such as 60%/40%,70%/30%, 80%/20%, 90%/10%, 95%/5%, 100%/0%, 40%/60%, 30%/70%, 20%/80%,10%/90% of bioderived/petroleum derived precursors, so long as at leasta portion of the product comprises a bioderived product produced by themicrobial organisms disclosed herein. It is understood that methods forproducing pharmaceutical formulations, humectants, solvents, sweeteners,preservative, food additives, monoglycerides, diglycerides, emulsifiers,antifreeze and de-icer agents, oil dispersants, solvents, resins,polyglycerol esters, moisturizer, oils, shortenings, margarines, andmedical, personal care, cosmetic or pharmaceutical preparations, and thelike, using the bioderived 1,2-propanediol, n-propanol, 1,3-propanediolor glycerol, or a bioderived 1,2-propanediol, n-propanol,1,3-propanediol or glycerol intermediate thereof, of the invention arewell known in the art.

In one embodiment, the product is a pharmaceutical formulation. In oneembodiment, the product is a humectant. In one embodiment, the productis a solvent, sweetener. In one embodiment, the product is apreservative. In one embodiment, the product is a food additive. In oneembodiment, the product is a monoglyceride. In one embodiment, theproduct is a diglyceride. In one embodiment, the product is aemulsifier. In one embodiment, the product is a antifreeze agent. In oneembodiment, the product is a de-icer agent. In one embodiment, theproduct is a oil dispersant. In one embodiment, the product is asolvent. In one embodiment, the product is a resin. In one embodiment,the product is a polyglycerol ester. In one embodiment, the product is amoisturizer. In one embodiment, the product is a oil. In one embodiment,the product is a shortening. In one embodiment, the product is amargarine. In one embodiment, the product is a medical preparation. Inone embodiment, the product is a personal care preparation. In oneembodiment, the product is a cosmetic preparation. In one embodiment,the product is a pharmaceutical preparation.

In some embodiments, provided herein is a culture medium comprisingbioderived 1,2-propanediol. In some embodiments, the bioderived1,2-propanediol is produced by culturing a non-naturally occurringmicrobial organism having a methanol metabolic pathway and1,2-propanediol pathway, as provided herein. In certain embodiments, thebioderived 1,2-propanediol has a carbon-12, carbon-13 and carbon-14isotope ratio that reflects an atmospheric carbon dioxide uptake source.In one embodiment, the culture medium is separated from a non-naturallyoccurring microbial organism having a methanol metabolic pathway and1,2-propanediol pathway.

In other embodiments, provided herein is a bioderived 1,2-propanediol.In some embodiments, the bioderived 1,2-propanediol is produced byculturing a non-naturally occurring microbial organism having a methanolmetabolic pathway and 1,2-propanediol pathway, as provided herein. Incertain embodiments, the bioderived 1,2-propanediol has a carbon-12,carbon-13 and carbon-14 isotope ratio that reflects an atmosphericcarbon dioxide uptake source. In some embodiments, the bioderived1,2-propanediol has an Fm value of at least 80%, at least 85%, at least90%, at least 95% or at least 98%. In certain embodiments, thebioderived 1,2-propanediol is a component of culture medium.

In certain embodiments, provided herein is a composition comprising abioderived 1,2-propanediol provided herein, for example, a bioderived1,2-propanediol produced by culturing a non-naturally occurringmicrobial organism having a methanol metabolic pathway and1,2-propanediol pathway, as provided herein. In some embodiments, thecomposition further comprises a compound other than said bioderived1,2-propanediol. In certain embodiments, the compound other than saidbioderived 1,2-propanediol is a trace amount of a cellular portion of anon-naturally occurring microbial organism having a methanol metabolicpathway and a 1,2-propanediol pathway, as provided herein.

In some embodiments, provided herein is a biobased product comprising abioderived 1,2-propanediol provided herein. In certain embodiments, thebiobased product is a pharmaceutical formulation, humectant, solvent,sweetener, preservative, food additive, monoglyceride, diglyceride,emulsifier, antifreeze agent, de-icer agent, oil dispersant, solvent,resin, polyglycerol ester, moisturizer, oil, shortening, margarine,medical preparation, personal care preparation, cosmetic preparation orpharmaceutical preparation. In certain embodiments, the biobased productcomprises at least 5% bioderived 1,2-propanediol. In certainembodiments, the biobased product comprises at least 10% bioderived1,2-propanediol. In some embodiments, the biobased product comprises atleast 20% bioderived 1,2-propanediol. In other embodiments, the biobasedproduct comprises at least 30% bioderived 1,2-propanediol. In someembodiments, the biobased product comprises at least 40% bioderived1,2-propanediol. In other embodiments, the biobased product comprises atleast 50% bioderived 1,2-propanediol. In one embodiment, the biobasedproduct comprises a portion of said bioderived 1,2-propanediol as arepeating unit. In another embodiment, provided herein is a moldedproduct obtained by molding the biobased product provided herein. Inother embodiments, provided herein is a process for producing a biobasedproduct provided herein, comprising chemically reacting said bioderived1,2-propanediol with itself or another compound in a reaction thatproduces said biobased product. In certain embodiments, provided hereinis a polymer comprising or obtained by converting the bioderived1,2-propanediol. In other embodiments, provided herein is a method forproducing a polymer, comprising chemically of enzymatically convertingthe bioderived 1,2-propanediol to the polymer. In yet other embodiments,provided herein is a composition comprising the bioderived1,2-propanediol, or a cell lysate or culture supernatant thereof.

In some embodiments, provided herein is a culture medium comprisingbioderived n-propanol. In some embodiments, the bioderived n-propanol isproduced by culturing a non-naturally occurring microbial organismhaving a methanol metabolic pathway and n-propanol pathway, as providedherein. In certain embodiments, the bioderived n-propanol has acarbon-12, carbon-13 and carbon-14 isotope ratio that reflects anatmospheric carbon dioxide uptake source. In one embodiment, the culturemedium is separated from a non-naturally occurring microbial organismhaving a methanol metabolic pathway and n-propanol pathway.

In other embodiments, provided herein is a bioderived n-propanol. Insome embodiments, the bioderived n-propanol is produced by culturing anon-naturally occurring microbial organism having a methanol metabolicpathway and n-propanol pathway, as provided herein. In certainembodiments, the bioderived n-propanol has a carbon-12, carbon-13 andcarbon-14 isotope ratio that reflects an atmospheric carbon dioxideuptake source. In some embodiments, the bioderived n-propanol has an Fmvalue of at least 80%, at least 85%, at least 90%, at least 95% or atleast 98%. In certain embodiments, the bioderived n-propanol is acomponent of culture medium.

In certain embodiments, provided herein is a composition comprising abioderived n-propanol provided herein, for example, a bioderivedn-propanol produced by culturing a non-naturally occurring microbialorganism having a methanol metabolic pathway and n-propanol pathway, asprovided herein. In some embodiments, the composition further comprisesa compound other than said bioderived n-propanol. In certainembodiments, the compound other than said bioderived n-propanol is atrace amount of a cellular portion of a non-naturally occurringmicrobial organism having a methanol metabolic pathway and a n-propanolpathway, as provided herein.

In some embodiments, provided herein is a biobased product comprising abioderived n-propanol provided herein. In certain embodiments, thebiobased product is a pharmaceutical formulation, humectant, solvent,sweetener, preservative, food additive, monoglyceride, diglyceride,emulsifier, antifreeze agent, de-icer agent, oil dispersant, solvent,resin, polyglycerol ester, moisturizer, oil, shortening, margarine,medical preparation, personal care preparation, cosmetic preparation orpharmaceutical preparation. In certain embodiments, the biobased productcomprises at least 5% bioderived n-propanol. In certain embodiments, thebiobased product comprises at least 10% bioderived n-propanol. In someembodiments, the biobased product comprises at least 20% bioderivedn-propanol. In other embodiments, the biobased product comprises atleast 30% bioderived n-propanol. In some embodiments, the biobasedproduct comprises at least 40% bioderived n-propanol. In otherembodiments, the biobased product comprises at least 50% bioderivedn-propanol. In one embodiment, the biobased product comprises a portionof said bioderived n-propanol as a repeating unit. In anotherembodiment, provided herein is a molded product obtained by molding thebiobased product provided herein. In other embodiments, provided hereinis a process for producing a biobased product provided herein,comprising chemically reacting said bioderived n-propanol with itself oranother compound in a reaction that produces said biobased product. Incertain embodiments, provided herein is a polymer comprising or obtainedby converting the bioderived n-propanol. In other embodiments, providedherein is a method for producing a polymer, comprising chemically ofenzymatically converting the bioderived n-propanol to the polymer. Inyet other embodiments, provided herein is a composition comprising thebioderived n-propanol, or a cell lysate or culture supernatant thereof.

In some embodiments, provided herein is a culture medium comprisingbioderived 1,3-propanediol. In some embodiments, the bioderived1,3-propanediol is produced by culturing a non-naturally occurringmicrobial organism having a methanol metabolic pathway and1,3-propanediol pathway, as provided herein. In certain embodiments, thebioderived 1,3-propanediol has a carbon-12, carbon-13 and carbon-14isotope ratio that reflects an atmospheric carbon dioxide uptake source.In one embodiment, the culture medium is separated from a non-naturallyoccurring microbial organism having a methanol metabolic pathway and1,3-propanediol pathway.

In other embodiments, provided herein is a bioderived 1,3-propanediol.In some embodiments, the bioderived 1,3-propanediol is produced byculturing a non-naturally occurring microbial organism having a methanolmetabolic pathway and 1,3-propanediol pathway, as provided herein. Incertain embodiments, the bioderived 1,3-propanediol has a carbon-12,carbon-13 and carbon-14 isotope ratio that reflects an atmosphericcarbon dioxide uptake source. In some embodiments, the bioderived1,3-propanediol has an Fm value of at least 80%, at least 85%, at least90%, at least 95% or at least 98%. In certain embodiments, thebioderived 1,3-propanediol is a component of culture medium.

In certain embodiments, provided herein is a composition comprising abioderived 1,3-propanediol, provided herein, for example, a bioderived1,3-propanediol produced by culturing a non-naturally occurringmicrobial organism having a methanol metabolic pathway and1,3-propanediol pathway, as provided herein. In some embodiments, thecomposition further comprises a compound other than said bioderived1,3-propanediol. In certain embodiments, the compound other than saidbioderived 1,3-propanediol is a trace amount of a cellular portion of anon-naturally occurring microbial organism having a methanol metabolicpathway and a 1,3-propanediol pathway, as provided herein.

In some embodiments, provided herein is a biobased product comprising abioderived 1,3-propanediol provided herein. In certain embodiments, thebiobased product is a pharmaceutical formulation, humectant, solvent,sweetener, preservative, food additive, monoglyceride, diglyceride,emulsifier, antifreeze agent, de-icer agent, oil dispersant, solvent,resin, polyglycerol ester, moisturizer, oil, shortening, margarine,medical preparation, personal care preparation, cosmetic preparation orpharmaceutical preparation. In certain embodiments, the biobased productcomprises at least 5% bioderived 1,3-propanediol. In certainembodiments, the biobased product comprises at least 10% bioderived1,3-propanediol. In some embodiments, the biobased product comprises atleast 20% bioderived 1,3-propanediol. In other embodiments, the biobasedproduct comprises at least 30% bioderived 1,3-propanediol. In someembodiments, the biobased product comprises at least 40% bioderived1,3-propanediol. In other embodiments, the biobased product comprises atleast 50% bioderived 1,3-propanediol. In one embodiment, the biobasedproduct comprises a portion of said bioderived 1,3-propanediol as arepeating unit. In another embodiment, provided herein is a moldedproduct obtained by molding the biobased product provided herein. Inother embodiments, provided herein is a process for producing a biobasedproduct provided herein, comprising chemically reacting said bioderived1,3-propanediol with itself or another compound in a reaction thatproduces said biobased product. In certain embodiments, provided hereinis a polymer comprising or obtained by converting the bioderived1,3-propanediol. In other embodiments, provided herein is a method forproducing a polymer, comprising chemically of enzymatically convertingthe bioderived 1,3-propanediol to the polymer. In yet other embodiments,provided herein is a composition comprising the bioderived1,3-propanediol, or a cell lysate or culture supernatant thereof.

In some embodiments, provided herein is a culture medium comprisingbioderived glycerol. In some embodiments, the bioderived glycerol isproduced by culturing a non-naturally occurring microbial organismhaving a methanol metabolic pathway and glycerol pathway, as providedherein. In certain embodiments, the bioderived glycerol has a carbon-12,carbon-13 and carbon-14 isotope ratio that reflects an atmosphericcarbon dioxide uptake source. In one embodiment, the culture medium isseparated from a non-naturally occurring microbial organism having amethanol metabolic pathway and glycerol pathway.

In other embodiments, provided herein is a bioderived glycerol. In someembodiments, the bioderived glycerol is produced by culturing anon-naturally occurring microbial organism having a methanol metabolicpathway and glycerol pathway, as provided herein. In certainembodiments, the bioderived glycerol has a carbon-12, carbon-13 andcarbon-14 isotope ratio that reflects an atmospheric carbon dioxideuptake source. In some embodiments, the bioderived glycerol has an Fmvalue of at least 80%, at least 85%, at least 90%, at least 95% or atleast 98%. In certain embodiments, the bioderived glycerol is acomponent of culture medium.

In certain embodiments, provided herein is a composition comprising abioderived glycerol provided herein, for example, a bioderived glycerolproduced by culturing a non-naturally occurring microbial organismhaving a methanol metabolic pathway and glycerol pathway, as providedherein. In some embodiments, the composition further comprises acompound other than said bioderived glycerol. In certain embodiments,the compound other than said bioderived glycerol is a trace amount of acellular portion of a non-naturally occurring microbial organism havinga methanol metabolic pathway and a glycerol pathway, as provided herein.

In some embodiments, provided herein is a biobased product comprising abioderived glycerol provided herein. In certain embodiments. thebiobased product is a pharmaceutical formulation, humectant, solvent,sweetener, preservative, food additive, monoglyceride, diglyceride,emulsifier, antifreeze agent, de-icer agent, oil dispersant, solvent,resin, polyglycerol ester, moisturizer, oil, shortening, margarine,medical preparation, personal care preparation, cosmetic preparation orpharmaceutical preparation. In certain embodiments, the biobased productcomprises at least 5% bioderived glycerol. In certain embodiments, thebiobased product comprises at least 10% bioderived glycerol. In someembodiments, the biobased product comprises at least 20% bioderivedglycerol. In other embodiments, the biobased product comprises at least30% bioderived glycerol. In some embodiments, the biobased productcomprises at least 40% bioderived glycerol. In other embodiments, thebiobased product comprises at least 50% bioderived glycerol. In oneembodiment, the biobased product comprises a portion of said bioderivedglycerol as a repeating unit. In another embodiment, provided herein isa molded product obtained by molding the biobased product providedherein. In other embodiments, provided herein is a process for producinga biobased product provided herein, comprising chemically reacting saidbioderived glycerol with itself or another compound in a reaction thatproduces said biobased product. In certain embodiments, provided hereinis a polymer comprising or obtained by converting the bioderivedglycerol. In other embodiments, provided herein is a method forproducing a polymer, comprising chemically of enzymatically convertingthe bioderived glycerol to the polymer. In yet other embodiments,provided herein is a composition comprising the bioderived glycerol, ora cell lysate or culture supernatant thereof.

The invention is described herein with general reference to themetabolic reaction, reactant or product thereof, or with specificreference to one or more nucleic acids or genes encoding an enzymeassociated with or catalyzing the referenced metabolic reaction,reactant or product. Unless otherwise expressly stated herein, thoseskilled in the art will understand that reference to a reaction alsoconstitutes reference to the reactants and products of the reaction.Similarly, unless otherwise expressly stated herein, reference to areactant or product also references the reaction and that reference toany of these metabolic constitutes also references the gene or genesencoding the enzymes that catalyze the referenced reaction, reactant orproduct. Likewise, given the well known fields of metabolicbiochemistry, enzymology and genomics, reference herein to a gene orencoding nucleic acid also constitutes a reference to the correspondingencoded enzyme and the reaction it catalyzes, or a protein associatedwith the reaction, as well as the reactants and products of thereaction.

Microbial organisms generally lack the capacity to synthesize1,2-propanediol, n-propanol, 1,3-propanediol and/or glycerol, andtherefore any of the compounds disclosed herein to be within the1,2-propanediol, n-propanol, 1,3-propanediol or glycerol family ofcompounds, or otherwise known by those in the art to be within the1,2-propanediol, n-propanol, 1,3-propanediol or glycerol family ofcompounds. Moreover, organisms having all of the requisite metabolicenzymatic capabilities are not known to produce 1,2-propanediol,n-propanol, 1,3-propanediol or glycerol from the enzymes described andbiochemical pathways exemplified herein. In contrast, the non-naturallyoccurring microbial organisms of the invention can generate1,2-propanediol, n-propanol, 1,3-propanediol or glycerol as a product,as well as intermediates thereof. The biosynthesis of 1,2-propanediol,n-propanol, 1,3-propanediol or glycerol, as well as intermediatesthereof, is particularly useful in chemical synthesis of1,2-propanediol, n-propanol, 1,3-propanediol or glycerol family ofcompounds, it also allows for the further biosynthesis of1,2-propanediol, n-propanol, 1,3-propanediol or glycerol familycompounds and avoids altogether chemical synthesis procedures.

The non-naturally occurring microbial organisms of the invention thatcan produce 1,2-propanediol, n-propanol, 1,3-propanediol or glycerol areproduced by ensuring that a host microbial organism includes functionalcapabilities for the complete biochemical synthesis of at least one1,2-propanediol, n-propanol, 1,3-propanediol or glycerol biosyntheticpathway of the invention. Ensuring at least one requisite1,2-propanediol, n-propanol, 1,3-propanediol or glycerol biosyntheticpathway confers 1,2-propanediol, n-propanol, 1,3-propanediol or glycerolbiosynthesis capability onto the host microbial organism.

The organisms and methods are described herein with general reference tothe metabolic reaction, reactant or product thereof, or with specificreference to one or more nucleic acids or genes encoding an enzymeassociated with or catalyzing, or a protein associated with, thereferenced metabolic reaction, reactant or product. Unless otherwiseexpressly stated herein, those skilled in the art will understand thatreference to a reaction also constitutes reference to the reactants andproducts of the reaction. Similarly, unless otherwise expressly statedherein, reference to a reactant or product also references the reaction,and reference to any of these metabolic constituents also references thegene or genes encoding the enzymes that catalyze or proteins involved inthe referenced reaction, reactant or product. Likewise, given the wellknown fields of metabolic biochemistry, enzymology and genomics,reference herein to a gene or encoding nucleic acid also constitutes areference to the corresponding encoded enzyme and the reaction itcatalyzes or a protein associated with the reaction as well as thereactants and products of the reaction.

The non-naturally occurring microbial organisms described herein can beproduced by introducing expressible nucleic acids encoding one or moreof the enzymes or proteins participating in one or more methanolmetabolic, formaldehyde assimilation, and/or 1,2-propanediol,n-propanol, 1,3-propanediol or glycerol biosynthetic pathways. Dependingon the host microbial organism chosen for biosynthesis, nucleic acidsfor some or all of a particular methanol metabolic, formaldehydeassimilation, and/or 1,2-propanediol, n-propanol, 1,3-propanediol orglycerol biosynthetic pathway can be expressed. For example, if a chosenhost is deficient in one or more enzymes or proteins for a desiredmetabolic, assimilation, or biosynthetic pathway, then expressiblenucleic acids for the deficient enzyme(s) or protein(s) are introducedinto the host for subsequent exogenous expression. Alternatively, if thechosen host exhibits endogenous expression of some pathway genes, but isdeficient in others, then an encoding nucleic acid is needed for thedeficient enzyme(s) or protein(s) to achieve 1,2-propanediol,n-propanol, 1,3-propanediol or glycerol biosynthesis and/or methanolmetabolism. Thus, a non-naturally occurring microbial organism describedherein can be produced by introducing exogenous enzyme or proteinactivities to obtain a desired metabolic pathway and/or a desiredbiosynthetic pathway can be obtained by introducing one or moreexogenous enzyme or protein activities that, together with one or moreendogenous enzymes or proteins, produces a desired product such as1,2-propanediol, n-propanol, 1,3-propanediol or glycerol.

Host microbial organisms can be selected from, and the non-naturallyoccurring microbial organisms generated in, for example, bacteria,yeast, fungus or any of a variety of other microorganisms applicable tofermentation processes. Exemplary bacteria include species selected fromEscherichia coli, Klebsiella oxytoca, Anaerobiospirillumsucciniciproducens, Actinobacillus succinogenes, Mannheimiasucciniciproducens, Rhizobium etli, Bacillus subtilis, Corynebacteriumglutamicum, Gluconobacter oxydans, Zymomonas mobilis, Lactococcuslactis, Lactobacillus plantarum, Streptomyces coelicolor, Clostridiumacetobutylicum, Pseudomonas fluorescens, and Pseudomonas putida.Exemplary yeasts or fungi include species selected from Saccharomycescerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis,Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger, Pichiapastoris, Rhizopus arrhizus, Rhizobus oryzae, and the like. E. coli is aparticularly useful host organism since it is a well characterizedmicrobial organism suitable for genetic engineering. Other particularlyuseful host organisms include yeast such as Saccharomyces cerevisiae. Itis understood that any suitable microbial host organism can be used tointroduce metabolic and/or genetic modifications to produce a desiredproduct.

Depending on the 1,2-propanediol, n-propanol, 1,3-propanediol orglycerol biosynthetic, methanol metabolic and/or formaldehydeassimilation pathway constituents of a selected host microbial organism,the non-naturally occurring microbial organisms provided herein willinclude at least one exogenously expressed 1,2-propanediol, n-propanol,1,3-propanediol or glycerol, formaldehyde assimilation and/or methanolmetabolic pathway-encoding nucleic acid and up to all encoding nucleicacids for one or more 1,2-propanediol, n-propanol, 1,3-propanediol orglycerol biosynthetic pathways, formaldehyde assimilation pathwaysand/or methanol metabolic pathways. For example, 1,2-propanediol,n-propanol, 1,3-propanediol or glycerol biosynthesis can be establishedin a host deficient in a pathway enzyme or protein through exogenousexpression of the corresponding encoding nucleic acid. In a hostdeficient in all enzymes or proteins of a 1,2-propanediol, n-propanol,1,3-propanediol or glycerol pathway, exogenous expression of all enzymeor proteins in the pathway can be included, although it is understoodthat all enzymes or proteins of a pathway can be expressed even if thehost contains at least one of the pathway enzymes or proteins. Forexample, exogenous expression of all enzymes or proteins in a pathwayfor production of 1,2-propanediol, n-propanol, 1,3-propanediol orglycerol can be included. The same holds true for the methanol metabolicpathways and formaldehyde assimilation pathways provided herein.

Given the teachings and guidance provided herein, those skilled in theart will understand that the number of encoding nucleic acids tointroduce in an expressible form will, at least, parallel the1,2-propanediol, n-propanol, 1,3-propanediol or glycerol pathway,formaldehyde assimilation pathway, and methanol metabolic pathwaydeficiencies of the selected host microbial organism. Therefore, anon-naturally occurring microbial organism of the invention can haveone, two, three, four, five, six, seven, eight, nine, or up to allnucleic acids encoding the enzymes or proteins constituting a methanolmetabolic pathway, formaldehyde assimilation pathway, and/or1,2-propanediol, n-propanol, 1,3-propanediol or glycerol biosyntheticpathway disclosed herein. In some embodiments, the non-naturallyoccurring microbial organisms also can include other geneticmodifications that facilitate or optimize 1,2-propanediol, n-propanol,1,3-propanediol or glycerol biosynthesis, formaldehyde assimilation,and/or methanol metabolism or that confer other useful functions ontothe host microbial organism. One such other functionality can include,for example, augmentation of the synthesis of one or more of the1,2-propanediol, n-propanol, 1,3-propanediol or glycerol pathwayprecursors.

Generally, a host microbial organism is selected such that it producesthe precursor of a 1,2-propanediol, n-propanol, 1,3-propanediol orglycerol pathway, either as a naturally produced molecule or as anengineered product that either provides de novo production of a desiredprecursor or increased production of a precursor naturally produced bythe host microbial organism. A host organism can be engineered toincrease production of a precursor, as disclosed herein. In addition, amicrobial organism that has been engineered to produce a desiredprecursor can be used as a host organism and further engineered toexpress enzymes or proteins of a 1,2-propanediol, n-propanol,1,3-propanediol or glycerol pathway.

In some embodiments, a non-naturally occurring microbial organismprovided herein is generated from a host that contains the enzymaticcapability to synthesize 1,2-propanediol, n-propanol, 1,3-propanediol orglycerol, assimilate formaldehyde and/or metabolize methanol. In thisspecific embodiment it can be useful to increase the synthesis oraccumulation of a 1,2-propanediol, n-propanol, 1,3-propanediol orglycerol pathway product, formaldehyde assimilation pathway productand/or methanol metabolic pathway product (e.g., reducing equivalentsand/or formaldehyde) to, for example, drive 1,2-propanediol, n-propanol,1,3-propanediol or glycerol pathway reactions toward 1,2-propanediol,n-propanol, 1,3-propanediol or glycerol production. Increased synthesisor accumulation can be accomplished by, for example, overexpression ofnucleic acids encoding one or more of the above-described1,2-propanediol, n-propanol, 1,3-propanediol or glycerol, formaldehydeassimilation and/or methanol metabolic pathway enzymes or proteins. Overexpression the enzyme(s) and/or protein(s) of the 1,2-propanediol,n-propanol, 1,3-propanediol or glycerol pathway, formaldehydeassimilation, and/or methanol metabolic pathway can occur, for example,through exogenous expression of the endogenous gene(s), or throughexogenous expression of the heterologous gene(s). Therefore, naturallyoccurring organisms can be readily generated to be non-naturallyoccurring microbial organisms, for example, producing 1,2-propanediol,n-propanol, 1,3-propanediol or glycerol through overexpression of one,two, three, four, five, six, seven, eight, up to all nucleic acidsencoding 1,2-propanediol, n-propanol, 1,3-propanediol or glycerolbiosynthetic pathway, and/or methanol metabolic pathway enzymes orproteins. Naturally occurring organisms can also be readily generated tobe non-naturally occurring microbial organisms, for example,assimilating formaldehyde, through overexpression of one, two, three,four, five, six, seven, eight, up to all nucleic acids encodingformaldehyde assimilation pathway, and/or methanol metabolic pathwayenzymes or proteins. In addition, a non-naturally occurring organism canbe generated by mutagenesis of an endogenous gene that results in anincrease in activity of an enzyme in the 1,2-propanediol, n-propanol,1,3-propanediol or glycerol, formaldehyde assimilation and/or methanolmetabolic pathway biosynthetic pathway.

In particularly useful embodiments, exogenous expression of the encodingnucleic acids is employed. Exogenous expression confers the ability tocustom tailor the expression and/or regulatory elements to the host andapplication to achieve a desired expression level that is controlled bythe user. However, endogenous expression also can be utilized in otherembodiments such as by removing a negative regulatory effector orinduction of the gene's promoter when linked to an inducible promoter orother regulatory element. Thus, an endogenous gene having a naturallyoccurring inducible promoter can be up-regulated by providing theappropriate inducing agent, or the regulatory region of an endogenousgene can be engineered to incorporate an inducible regulatory element,thereby allowing the regulation of increased expression of an endogenousgene at a desired time. Similarly, an inducible promoter can be includedas a regulatory element for an exogenous gene introduced into anon-naturally occurring microbial organism.

It is understood that, in methods provided herein, any of the one ormore exogenous nucleic acids can be introduced into a microbial organismto produce a non-naturally occurring microbial organism provided herein.The nucleic acids can be introduced so as to confer, for example, a3-hydroxyisobutyrate or MAA biosynthetic, formaldehyde assimilationand/or methanol metabolic pathway onto the microbial organism.Alternatively, encoding nucleic acids can be introduced to produce anintermediate microbial organism having the biosynthetic capability tocatalyze some of the required reactions to confer 3-hydroxyisobutyrateor MAA biosynthetic, formaldehyde assimilation and/or methanol metaboliccapability. For example, a non-naturally occurring microbial organismhaving a 3-hydroxyisobutyrate or MAA biosynthetic pathway, formaldehydeassimilation pathway and/or methanol metabolic pathway can comprise atleast two exogenous nucleic acids encoding desired enzymes or proteins.Thus, it is understood that any combination of two or more enzymes orproteins of a biosynthetic pathway, formaldehyde assimilation pathwayand/or metabolic pathway can be included in a non-naturally occurringmicrobial organism provided herein. Similarly, it is understood that anycombination of three or more enzymes or proteins of a biosyntheticpathway, formaldehyde assimilation pathway and/or metabolic pathway canbe included in a non-naturally occurring microbial organism of theinvention, as desired, so long as the combination of enzymes and/orproteins of the desired biosynthetic pathway, formaldehyde assimilationpathway and/or metabolic pathway results in production of thecorresponding desired product. Similarly, any combination of four ormore enzymes or proteins of a biosynthetic pathway, formaldehydeassimilation pathway and/or methanol metabolic pathway as disclosedherein can be included in a non-naturally occurring microbial organismprovided herein, as desired, so long as the combination of enzymesand/or proteins of the desired biosynthetic, assimilation and/ormetabolic pathway results in production of the corresponding desiredproduct.

In addition to the metabolism of methanol, assimilation of formaldehyde,and biosynthesis of 1,2-propanediol, n-propanol, 1,3-propanediol orglycerol, as described herein, the non-naturally occurring microbialorganisms and methods provided also can be utilized in variouscombinations with each other and with other microbial organisms andmethods well known in the art to achieve product biosynthesis by otherroutes. For example, one alternative produce 1,2-propanediol,n-propanol, 1,3-propanediol or glycerol, other than use of the1,2-propanediol, n-propanol, 1,3-propanediol or glycerol producers isthrough addition of another microbial organism capable of converting a1,2-propanediol, n-propanol, 1,3-propanediol or glycerol pathwayintermediate to 1,2-propanediol, n-propanol, 1,3-propanediol orglycerol. One such procedure includes, for example, the fermentation ofa microbial organism that produces a 1,2-propanediol, n-propanol,1,3-propanediol or glycerol pathway intermediate. The 1,2-propanediol,n-propanol, 1,3-propanediol or glycerol pathway intermediate can then beused as a substrate for a second microbial organism that converts the1,2-propanediol, n-propanol, 1,3-propanediol or glycerol pathwayintermediate to 1,2-propanediol, n-propanol, 1,3-propanediol orglycerol. The 1,2-propanediol, n-propanol, 1,3-propanediol or glycerolpathway intermediate can be added directly to another culture of thesecond organism or the original culture of the 1,2-propanediol,n-propanol, 1,3-propanediol or glycerol pathway intermediate producerscan be depleted of these microbial organisms by, for example, cellseparation, and then subsequent addition of the second organism to thefermentation broth can be utilized to produce the final product withoutintermediate purification steps.

In other embodiments, the non-naturally occurring microbial organismsand methods of the invention can be assembled in a wide variety ofsubpathways to achieve biosynthesis of for example, 1,2-propanediol,n-propanol, 1,3-propanediol or glycerol. In these embodiments,biosynthetic pathways for a desired product can be segregated intodifferent microbial organisms, and the different microbial organisms canbe co-cultured to produce the final product. In such a biosyntheticscheme, the product of one microbial organism is the substrate for asecond microbial organism until the final product is synthesized. Forexample, the biosynthesis of 1,2-propanediol, n-propanol,1,3-propanediol or glycerol can be accomplished by constructing amicrobial organism that contains biosynthetic pathways for conversion ofone pathway intermediate to another pathway intermediate or the product.Alternatively, 1,2-propanediol, n-propanol, 1,3-propanediol or glycerolalso can be biosynthetically produced from microbial organisms throughco-culture or co-fermentation using two organisms in the same vessel,where the first microbial organism produces a 1,2-propanediol,n-propanol, 1,3-propanediol or glycerol intermediate and the secondmicrobial organism converts the intermediate to 1,2-propanediol,n-propanol, 1,3-propanediol or glycerol.

Given the teachings and guidance provided herein, those skilled in theart will understand that a wide variety of combinations and permutationsexist for the non-naturally occurring microbial organisms and methodstogether with other microbial organisms, with the co-culture of othernon-naturally occurring microbial organisms having subpathways and withcombinations of other chemical and/or biochemical procedures well knownin the art to produce 1,2-propanediol, n-propanol, 1,3-propanediol orglycerol and/or metabolize methanol.

Sources of encoding nucleic acids for a 1,2-propanediol, n-propanol,1,3-propanediol or glycerol, formaldehyde assimilation, or methanolmetabolic pathway enzyme or protein can include, for example, anyspecies where the encoded gene product is capable of catalyzing thereferenced reaction. Such species include both prokaryotic andeukaryotic organisms including, but not limited to, bacteria, includingarchaea and eubacteria, and eukaryotes, including yeast, plant, insect,animal, and mammal, including human. Exemplary species for such sourcesinclude, for example, Escherichia coli, Saccharomyces cerevisiae,Saccharomyces kluyveri, Candida boidinii, Clostridium kluyveri,Clostridium acetobutylicum, Clostridium beijerinckii, Clostridiumsaccharoperbutylacetonicum, Clostridium perfringens, Clostridiumdifficile, Clostridium botulinum, Clostridium tyrobutyricum, Clostridiumtetanomorphum, Clostridium tetani, Clostridium propionicum, Clostridiumaminobutyricum, Clostridium subterminale, Clostridium sticklandii,Ralstonia eutropha, Mycobacterium Bovis, Mycobacterium tuberculosis,Porphyromonas gingivalis, Arabidopsis thaliana, Thermus thermophilus,Pseudomonas species, including Pseudomonas aeruginosa, Pseudomonasputida, Pseudomonas stutzeri, Pseudomonas fluorescens, Homo sapiens,Oryctolagus cuniculus, Rhodobacter spaeroides, Thermoanaerobacterbrockii, Metallosphaera sedula, Leuconostoc mesenteroides, Chloroflexusaurantiacus, Roseiflexus castenholzii, Erythrobacter, Simmondsiachinensis, Acinetobacter species, including Acinetobacter calcoaceticusand Acinetobacter baylyi, Porphyromonas gingivalis, Sulfolobus tokodaii,Sulfolobus solfataricus, Sulfolobus acidocaldarius, Bacillus subtilis,Bacillus cereus, Bacillus megaterium, Bacillus brevis, Bacillus pumilus,Rattus norvegicus, Klebsiella pneumonia, Klebsiella oxytoca, Euglenagracilis, Treponema denticola, Moorella thermoacetica, Thermotogamaritima, Halobacterium salinarum, Geobacillus stearothermophilus,Aeropyrum pernix, Sus scrofa, Caenorhabditis elegans, Corynebacteriumglutamicum, Acidaminococcus fermentans, Lactococcus lactis,Lactobacillus plantarum, Streptococcus thermophilus, Enterobacteraerogenes, Candida, Aspergillus terreus, Pedicoccus pentosaceus,Zymomonas mobilus, Acetobacter pasteurians, Kluyveromyces lactis,Eubacterium barkeri, Bacteroides capillosus, Anaerotruncus colihominis,Natranaerobius thermophilusm, Campylobacter jejuni, Haemophilusinfluenzae, Serratia marcescens, Citrobacter amalonaticus, Myxococcusxanthus, Fusobacterium nuleatum, Penicillium chrysogenum, marine gammaproteobacierium, butyrate producingbacterium, Nocardia iowensis,Nocardia jarcinica, Streptomyces griseus, Schizosaccharomyces pombe,Geobacillus thermoglucosidasius, Salmonella typhimurium, Vibrio cholera,Heliobacter pylori, Nicotiana tabacum, Oryza sativa, Haloferaxmediterranei, Agrobacterium tumefaciens, Achromobacter denitrificans,Fusobacterium nucleatum, Streptomyces clavuligenus, Acinetobacterbaumanii, Mus musculus, Lachancea kluyveri, Trichomonas vaginalis,Trypanosoma brucei, Pseudomonas stutzeri, Bradyrhizobium japonicum,Mesorhizobium loti, Bos taurus, Nicotiana glutinosa, Vibrio vulnificus,Selenomonas ruminantium, Vibrio parahaemolyticus, Archaeoglobusfulgidus, Haloarcula marismortui, Pyrobaculum aerophilum, Mycobacteriumsmegmatis MC2 155, Mycobacterium avium subsp. paratuberculosis K-10,Mycobacterium marinum M, Tsukamurella paurometabola DSM 20162, CyanobiumPCC7001, Dictyostelium discoideum AX-1, as well as other exemplaryspecies disclosed herein or available as source organisms forcorresponding genes. However, with the complete genome sequenceavailable for now more than 550 species (with more than half of theseavailable on public databases such as the NCBI), including 395microorganism genomes and a variety of yeast, fungi, plant, andmammalian genomes, the identification of genes encoding the requisite1,2-propanediol, n-propanol, 1,3-propanediol or glycerol biosyntheticactivity for one or more genes in related or distant species, includingfor example, homologues, orthologs, paralogs and nonorthologous genedisplacements of known genes, and the interchange of genetic alterationsbetween organisms is routine and well known in the art. Accordingly, themetabolic alterations allowing biosynthesis of 1,2-propanediol,n-propanol, 1,3-propanediol or glycerol, metabolism of methanol and/orassimilation of formaldehyde described herein with reference to aparticular organism such as E. coli can be readily applied to othermicroorganisms, including prokaryotic and eukaryotic organisms alike.Given the teachings and guidance provided herein, those skilled in theart will know that a metabolic alteration exemplified in one organismcan be applied equally to other organisms.

In some instances, such as when an alternative 1,2-propanediol,n-propanol, 1,3-propanediol or glycerol biosynthetic, formaldehydeassimilation and/or methanol metabolic pathway exists in an unrelatedspecies, 1,2-propanediol, n-propanol, 1,3-propanediol or glycerolbiosynthesis, formaldehyde assimilation and/or methanol metabolism canbe conferred onto the host species by, for example, exogenous expressionof a paralog or paralogs from the unrelated species that catalyzes asimilar, yet non-identical metabolic reaction to replace the referencedreaction. Because certain differences among metabolic networks existbetween different organisms, those skilled in the art will understandthat the actual gene usage between different organisms may differ.However, given the teachings and guidance provided herein, those skilledin the art also will understand that the teachings and methods providedherein can be applied to all microbial organisms using the cognatemetabolic alterations to those exemplified herein to construct amicrobial organism in a species of interest that will synthesize1,2-propanediol, n-propanol, 1,3-propanediol or glycerol, assimilateformaldehyde, and/or metabolize methanol.

Methods for constructing and testing the expression levels of anon-naturally occurring 1,2-propanediol-, n-propanol-, 1,3-propanediol-or glycerol-producing host can be performed, for example, by recombinantand detection methods well known in the art. Such methods can be founddescribed in, for example, Sambrook et al., Molecular Cloning: ALaboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York(2001); and Ausubel et al., Current Protocols in Molecular Biology, JohnWiley and Sons, Baltimore, Md. (1999).

Exogenous nucleic acid sequences involved in a pathway for metabolism ofmethanol, assimilation of formaldehyde and/or production of1,2-propanediol, n-propanol, 1,3-propanediol or glycerol can beintroduced stably or transiently into a host cell using techniques wellknown in the art including, but not limited to, conjugation,electroporation, chemical transformation, transduction, transfection,and ultrasound transformation. For exogenous expression in E. coli orother prokaryotic cells, some nucleic acid sequences in the genes orcDNAs of eukaryotic nucleic acids can encode targeting signals such asan N-terminal mitochondrial or other targeting signal, which can beremoved before transformation into prokaryotic host cells, if desired.For example, removal of a mitochondrial leader sequence led to increasedexpression in E. coli (Hoffmeister et al., J. Biol. Chem. 280:4329-4338(2005)). For exogenous expression in yeast or other eukaryotic cells,genes can be expressed in the cytosol without the addition of leadersequence, or can be targeted to mitochondrion or other organelles, ortargeted for secretion, by the addition of a suitable targeting sequencesuch as a mitochondrial targeting or secretion signal suitable for thehost cells. Thus, it is understood that appropriate modifications to anucleic acid sequence to remove or include a targeting sequence can beincorporated into an exogenous nucleic acid sequence to impart desirableproperties. Furthermore, genes can be subjected to codon optimizationwith techniques well known in the art to achieve optimized expression ofthe proteins.

An expression vector or vectors can be constructed to include one ormore 1,2-propanediol, n-propanol, 1,3-propanediol or glycerolbiosynthetic, formaldehyde assimilation and/or methanol metabolicpathway encoding nucleic acids as exemplified herein operably linked toexpression control sequences functional in the host organism. Expressionvectors applicable for use in the microbial host organisms providedinclude, for example, plasmids, phage vectors, viral vectors, episomesand artificial chromosomes, including vectors and selection sequences ormarkers operable for stable integration into a host chromosome.Additionally, the expression vectors can include one or more selectablemarker genes and appropriate expression control sequences. Selectablemarker genes also can be included that, for example, provide resistanceto antibiotics or toxins, complement auxotrophic deficiencies, or supplycritical nutrients not in the culture media. Expression controlsequences can include constitutive and inducible promoters,transcription enhancers, transcription terminators, and the like whichare well known in the art. When two or more exogenous encoding nucleicacids are to be co-expressed, both nucleic acids can be inserted, forexample, into a single expression vector or in separate expressionvectors. For single vector expression, the encoding nucleic acids can beoperationally linked to one common expression control sequence or linkedto different expression control sequences, such as one induciblepromoter and one constitutive promoter. The transformation of exogenousnucleic acid sequences involved in a metabolic or synthetic pathway canbe confirmed using methods well known in the art. Such methods include,for example, nucleic acid analysis such as Northern blots or polymerasechain reaction (PCR) amplification of mRNA, or immunoblotting forexpression of gene products, or other suitable analytical methods totest the expression of an introduced nucleic acid sequence or itscorresponding gene product. It is understood by those skilled in the artthat the exogenous nucleic acid is expressed in a sufficient amount toproduce the desired product, and it is further understood thatexpression levels can be optimized to obtain sufficient expression usingmethods well known in the art and as disclosed herein.

Suitable purification and/or assays to test, e.g., for the production of1,2-propanediol, n-propanol, 1,3-propanediol or glycerol can beperformed using well known methods. Suitable replicates such astriplicate cultures can be grown for each engineered strain to betested. For example, product and byproduct formation in the engineeredproduction host can be monitored. The final product and intermediates,and other organic compounds, can be analyzed by methods such as HPLC(High Performance Liquid Chromatography), GC-MS (Gas Chromatography-MassSpectroscopy) and LC-MS (Liquid Chromatography-Mass Spectroscopy) orother suitable analytical methods using routine procedures well known inthe art. The release of product in the fermentation broth can also betested with the culture supernatant. Byproducts and residual glucose canbe quantified by HPLC using, for example, a refractive index detectorfor glucose and alcohols, and a UV detector for organic acids (Lin etal., Biotechnol. Bioeng. 90:775-779 (2005)), or other suitable assay anddetection methods well known in the art. The individual enzyme orprotein activities from the exogenous DNA sequences can also be assayedusing methods well known in the art.

The 1,2-propanediol, n-propanol, 1,3-propanediol or glycerol can beseparated from other components in the culture using a variety ofmethods well known in the art. Such separation methods include, forexample, extraction procedures as well as methods that includecontinuous liquid-liquid extraction, pervaporation, membrane filtration,membrane separation, reverse osmosis, electrodialysis, distillation,crystallization, centrifugation, extractive filtration, ion exchangechromatography, size exclusion chromatography, adsorptionchromatography, and ultrafiltration. All of the above methods are wellknown in the art.

Any of the non-naturally occurring microbial organisms described hereincan be cultured to produce and/or secrete the biosynthetic products, orintermediates thereof. For example, the 1,2-propanediol, n-propanol,1,3-propanediol or glycerol producers can be cultured for thebiosynthetic production of 1,2-propanediol, n-propanol, 1,3-propanediolor glycerol. Accordingly, in some embodiments, the invention providesculture medium having a 1,2-propanediol, n-propanol, 1,3-propanediol orglycerol, formaldehyde assimilation and/or methanol metabolic pathwayintermediate described herein. In some aspects, the culture medium canalso be separated from the non-naturally occurring microbial organismsprovided herein that produced the 1,2-propanediol, n-propanol,1,3-propanediol or glycerol, formaldehyde assimilation and/or methanolmetabolic pathway intermediate. Methods for separating a microbialorganism from culture medium are well known in the art. Exemplarymethods include filtration, flocculation, precipitation, centrifugation,sedimentation, and the like.

In certain embodiments, for example, for the production of theproduction of 1,2-propanediol, n-propanol, 1,3-propanediol or glycerol,the recombinant strains are cultured in a medium with carbon source andother essential nutrients. It is sometimes desirable and can be highlydesirable to maintain anaerobic conditions in the fermenter to reducethe cost of the overall process. Such conditions can be obtained, forexample, by first sparging the medium with nitrogen and then sealing theflasks with a septum and crimp-cap. For strains where growth is notobserved anaerobically, microaerobic or substantially anaerobicconditions can be applied by perforating the septum with a small holefor limited aeration. Exemplary anaerobic conditions have been describedpreviously and are well-known in the art. Exemplary aerobic andanaerobic conditions are described, for example, in U.S. Publ. No.2009/0047719. Fermentations can be performed in a batch, fed-batch orcontinuous manner, as disclosed herein. Fermentations can also beconducted in two phases, if desired. The first phase can be aerobic toallow for high growth and therefore high productivity, followed by ananaerobic phase of high 1,2-propanediol, n-propanol, 1,3-propanediol orglycerol yields.

If desired, the pH of the medium can be maintained at a desired pH, inparticular neutral pH, such as a pH of around 7 by addition of a base,such as NaOH or other bases, or acid, as needed to maintain the culturemedium at a desirable pH. The growth rate can be determined by measuringoptical density using a spectrophotometer (600 nm), and the glucoseuptake rate by monitoring carbon source depletion over time.

The growth medium, can include, for example, any carbohydrate sourcewhich can supply a source of carbon to the non-naturally occurringmicroorganism. Such sources include, for example, sugars such asglucose, xylose, arabinose, galactose, mannose, fructose, sucrose andstarch; or glycerol, alone as the sole source of carbon or incombination with other carbon sources described herein or known in theart. In one embodiment, the carbon source is a sugar. In one embodiment,the carbon source is a sugar-containing biomass. In some embodiments,the sugar is glucose. In one embodiment, the sugar is xylose. In anotherembodiment, the sugar is arabinose. In one embodiment, the sugar isgalactose. In another embodiment, the sugar is fructose. In otherembodiments, the sugar is sucrose. In one embodiment, the sugar isstarch. In certain embodiments, the carbon source is glycerol. In someembodiments, the carbon source is crude glycerol. In one embodiment, thecarbon source is crude glycerol without treatment. In other embodiments,the carbon source is glycerol and glucose. In another embodiment, thecarbon source is methanol and glycerol. In one embodiment, the carbonsource is carbon dioxide. In one embodiment, the carbon source isformate. In one embodiment, the carbon source is methane. In oneembodiment, the carbon source is methanol. In one embodiment, the carbonsource is chemoelectro-generated carbon (see, e.g., Liao et al. (2012)Science 335:1596). In one embodiment, the chemoelectro-generated carbonis methanol. In one embodiment, the chemoelectro-generated carbon isformate. In one embodiment, the chemoelectro-generated carbon is formateand methanol. In one embodiment, the carbon source is a sugar andmethanol. In another embodiment, the carbon source is a sugar andglycerol. In other embodiments, the carbon source is a sugar and crudeglycerol. In yet other embodiments, the carbon source is a sugar andcrude glycerol without treatment. In one embodiment, the carbon sourceis a sugar-containing biomass and methanol. In another embodiment, thecarbon source is a sugar-containing biomass and glycerol. In otherembodiments, the carbon source is a sugar-containing biomass and crudeglycerol. In yet other embodiments, the carbon source is asugar-containing biomass and crude glycerol without treatment. Othersources of carbohydrate include, for example, renewable feedstocks andbiomass. Exemplary types of biomasses that can be used as feedstocks inthe methods of the invention include cellulosic biomass, hemicellulosicbiomass and lignin feedstocks or portions of feedstocks. Such biomassfeedstocks contain, for example, carbohydrate substrates useful ascarbon sources such as glucose, xylose, arabinose, galactose, mannose,fructose and starch. Given the teachings and guidance provided herein,those skilled in the art will understand that renewable feedstocks andbiomass other than those exemplified above also can be used forculturing the microbial organisms provided herein for the production of1,2-propanediol, n-propanol, 1,3-propanediol or glycerol, and otherpathway intermediates.

In one embodiment, the carbon source is glycerol. In certainembodiments, the glycerol carbon source is crude glycerol or crudeglycerol without further treatment. In a further embodiment, the carbonsource comprises glycerol or crude glycerol, and also sugar or asugar-containing biomass, such as glucose. In a specific embodiment, theconcentration of glycerol in the fermentation broth is maintained byfeeding crude glycerol, or a mixture of crude glycerol and sugar (e.g.,glucose). In certain embodiments, sugar is provided for sufficientstrain growth. In some embodiments, the sugar (e.g., glucose) isprovided at a molar concentration ratio of glycerol to sugar of from200:1 to 1:200. In some embodiments, the sugar (e.g., glucose) isprovided at a molar concentration ratio of glycerol to sugar of from100:1 to 1:100. In some embodiments, the sugar (e.g., glucose) isprovided at a molar concentration ratio of glycerol to sugar of from100:1 to 5:1. In some embodiments, the sugar (e.g., glucose) is providedat a molar concentration ratio of glycerol to sugar of from 50:1 to 5:1.In certain embodiments, the sugar (e.g., glucose) is provided at a molarconcentration ratio of glycerol to sugar of 100:1. In one embodiment,the sugar (e.g., glucose) is provided at a molar concentration ratio ofglycerol to sugar of 90:1. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of glycerol to sugar of 80:1.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of glycerol to sugar of 70:1. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio ofglycerol to sugar of 60:1. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of glycerol to sugar of 50:1.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of glycerol to sugar of 40:1. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio ofglycerol to sugar of 30:1. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of glycerol to sugar of 20:1.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of glycerol to sugar of 10:1. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio ofglycerol to sugar of 5:1. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of glycerol to sugar of 2:1.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of glycerol to sugar of 1:1. In certain embodiments,the sugar (e.g., glucose) is provided at a molar concentration ratio ofglycerol to sugar of 1:100. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of glycerol to sugar of 1:90.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of glycerol to sugar of 1:80. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio ofglycerol to sugar of 1:70. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of glycerol to sugar of 1:60.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of glycerol to sugar of 1:50. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio ofglycerol to sugar of 1:40. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of glycerol to sugar of 1:30.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of glycerol to sugar of 1:20. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio ofglycerol to sugar of 1:10. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of glycerol to sugar of 1:5.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of glycerol to sugar of 1:2. In certain embodimentsof the ratios provided above, the sugar is a sugar-containing biomass.In certain other embodiments of the ratios provided above, the glycerolis a crude glycerol or a crude glycerol without further treatment. Inother embodiments of the ratios provided above, the sugar is asugar-containing biomass, and the glycerol is a crude glycerol or acrude glycerol without further treatment.

Crude glycerol can be a by-product produced in the production ofbiodiesel, and can be used for fermentation without any furthertreatment. Biodiesel production methods include (1) a chemical methodwherein the glycerol-group of vegetable oils or animal oils issubstituted by low-carbon alcohols such as methanol or ethanol toproduce a corresponding fatty acid methyl esters or fatty acid ethylesters by transesterification in the presence of acidic or basiccatalysts; (2) a biological method where biological enzymes or cells areused to catalyze transesterification reaction and the correspondingfatty acid methyl esters or fatty acid ethyl esters are produced; and(3) a supercritical method, wherein transesterification reaction iscarried out in a supercritical solvent system without any catalysts. Thechemical composition of crude glycerol can vary with the process used toproduce biodiesel, the transesterification efficiency, recoveryefficiency of the biodiesel, other impurities in the feedstock, andwhether methanol and catalysts were recovered. For example, the chemicalcompositions of eleven crude glycerol collected from seven Australianbiodiesel producers reported that glycerol content ranged between 38%and 96%, with some samples including more than 14% methanol and 29% ash.In certain embodiments, the crude glycerol comprises from 5% to 99%glycerol. In some embodiments, the crude glycerol comprises from 10% to90% glycerol. In some embodiments, the crude glycerol comprises from 10%to 80% glycerol. In some embodiments, the crude glycerol comprises from10% to 70% glycerol. In some embodiments, the crude glycerol comprisesfrom 10% to 60% glycerol. In some embodiments, the crude glycerolcomprises from 10% to 50% glycerol. In some embodiments, the crudeglycerol comprises from 10% to 40% glycerol. In some embodiments, thecrude glycerol comprises from 10% to 30% glycerol. In some embodiments,the crude glycerol comprises from 10% to 20% glycerol. In someembodiments, the crude glycerol comprises from 80% to 90% glycerol. Insome embodiments, the crude glycerol comprises from 70% to 90% glycerol.In some embodiments, the crude glycerol comprises from 60% to 90%glycerol. In some embodiments, the crude glycerol comprises from 50% to90% glycerol. In some embodiments, the crude glycerol comprises from 40%to 90% glycerol. In some embodiments, the crude glycerol comprises from30% to 90% glycerol. In some embodiments, the crude glycerol comprisesfrom 20% to 90% glycerol. In some embodiments, the crude glycerolcomprises from 20% to 40% glycerol. In some embodiments, the crudeglycerol comprises from 40% to 60% glycerol. In some embodiments, thecrude glycerol comprises from 60% to 80% glycerol. In some embodiments,the crude glycerol comprises from 50% to 70% glycerol. In oneembodiment, the glycerol comprises 5% glycerol. In one embodiment, theglycerol comprises 10% glycerol. In one embodiment, the glycerolcomprises 15% glycerol. In one embodiment, the glycerol comprises 20%glycerol. In one embodiment, the glycerol comprises 25% glycerol. In oneembodiment, the glycerol comprises 30% glycerol. In one embodiment, theglycerol comprises 35% glycerol. In one embodiment, the glycerolcomprises 40% glycerol. In one embodiment, the glycerol comprises 45%glycerol. In one embodiment, the glycerol comprises 50% glycerol. In oneembodiment, the glycerol comprises 55% glycerol. In one embodiment, theglycerol comprises 60% glycerol. In one embodiment, the glycerolcomprises 65% glycerol. In one embodiment, the glycerol comprises 70%glycerol. In one embodiment, the glycerol comprises 75% glycerol. In oneembodiment, the glycerol comprises 80% glycerol. In one embodiment, theglycerol comprises 85% glycerol. In one embodiment, the glycerolcomprises 90% glycerol. In one embodiment, the glycerol comprises 95%glycerol. In one embodiment, the glycerol comprises 99% glycerol.

In one embodiment, the carbon source is methanol or formate. In certainembodiments, methanol is used as a carbon source in the formaldehydeassimilation pathways provided herein. In one embodiment, the carbonsource is methanol or formate. In other embodiments, formate is used asa carbon source in the formaldehyde assimilation pathways providedherein. In specific embodiments, methanol is used as a carbon source inthe methanol metabolic pathways provided herein, either alone or incombination with the product pathways provided herein.

In one embodiment, the carbon source comprises methanol, and sugar(e.g., glucose) or a sugar-containing biomass. In another embodiment,the carbon source comprises formate, and sugar (e.g., glucose) or asugar-containing biomass. In one embodiment, the carbon source comprisesmethanol, formate, and sugar (e.g., glucose) or a sugar-containingbiomass. In specific embodiments, the methanol or formate, or both, inthe fermentation feed is provided as a mixture with sugar (e.g.,glucose) or sugar-comprising biomass. In certain embodiments, sugar isprovided for sufficient strain growth.

In certain embodiments, the carbon source comprises methanol and a sugar(e.g., glucose). In some embodiments, the sugar (e.g., glucose) isprovided at a molar concentration ratio of methanol to sugar of from200:1 to 1:200. In some embodiments, the sugar (e.g., glucose) isprovided at a molar concentration ratio of methanol to sugar of from100:1 to 1:100. In some embodiments, the sugar (e.g., glucose) isprovided at a molar concentration ratio of methanol to sugar of from100:1 to 5:1. In some embodiments, the sugar (e.g., glucose) is providedat a molar concentration ratio of methanol to sugar of from 50:1 to 5:1.In certain embodiments, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol to sugar of 100:1. In one embodiment,the sugar (e.g., glucose) is provided at a molar concentration ratio ofmethanol to sugar of 90:1. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of methanol to sugar of 80:1.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol to sugar of 70:1. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio ofmethanol to sugar of 60:1. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of methanol to sugar of 50:1.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol to sugar of 40:1. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio ofmethanol to sugar of 30:1. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of methanol to sugar of 20:1.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol to sugar of 10:1. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio ofmethanol to sugar of 5:1. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of methanol to sugar of 2:1.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol to sugar of 1:1. In certain embodiments,the sugar (e.g., glucose) is provided at a molar concentration ratio ofmethanol to sugar of 1:100. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of methanol to sugar of 1:90.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol to sugar of 1:80. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio ofmethanol to sugar of 1:70. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of methanol to sugar of 1:60.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol to sugar of 1:50. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio ofmethanol to sugar of 1:40. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of methanol to sugar of 1:30.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol to sugar of 1:20. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio ofmethanol to sugar of 1:10. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of methanol to sugar of 1:5.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol to sugar of 1:2. In certain embodimentsof the ratios provided above, the sugar is a sugar-containing biomass.

In certain embodiments, the carbon source comprises formate and a sugar(e.g., glucose). In some embodiments, the sugar (e.g., glucose) isprovided at a molar concentration ratio of formate to sugar of from200:1 to 1:200. In some embodiments, the sugar (e.g., glucose) isprovided at a molar concentration ratio of formate to sugar of from100:1 to 1:100. In some embodiments, the sugar (e.g., glucose) isprovided at a molar concentration ratio of formate to sugar of from100:1 to 5:1. In some embodiments, the sugar (e.g., glucose) is providedat a molar concentration ratio of formate to sugar of from 50:1 to 5:1.In certain embodiments, the sugar (e.g., glucose) is provided at a molarconcentration ratio of formate to sugar of 100:1. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio offormate to sugar of 90:1. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of formate to sugar of 80:1.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of formate to sugar of 70:1. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio offormate to sugar of 60:1. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of formate to sugar of 50:1.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of formate to sugar of 40:1. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio offormate to sugar of 30:1. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of formate to sugar of 20:1.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of formate to sugar of 10:1. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio offormate to sugar of 5:1. In one embodiment, the sugar (e.g., glucose) isprovided at a molar concentration ratio of formate to sugar of 2:1. Inone embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of formate to sugar of 1:1. In certain embodiments,the sugar (e.g., glucose) is provided at a molar concentration ratio offormate to sugar of 1:100. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of formate to sugar of 1:90.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of formate to sugar of 1:80. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio offormate to sugar of 1:70. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of formate to sugar of 1:60.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of formate to sugar of 1:50. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio offormate to sugar of 1:40. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of formate to sugar of 1:30.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of formate to sugar of 1:20. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio offormate to sugar of 1:10. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of formate to sugar of 1:5.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of formate to sugar of 1:2. In certain embodimentsof the ratios provided above, the sugar is a sugar-containing biomass.

In certain embodiments, the carbon source comprises a mixture ofmethanol and formate, and a sugar (e.g., glucose). In certainembodiments, sugar is provided for sufficient strain growth. In someembodiments, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of from 200:1 to1:200. In some embodiments, the sugar (e.g., glucose) is provided at amolar concentration ratio of methanol and formate to sugar of from 100:1to 1:100. In some embodiments, the sugar (e.g., glucose) is provided ata molar concentration ratio of methanol and formate to sugar of from100:1 to 5:1. In some embodiments, the sugar (e.g., glucose) is providedat a molar concentration ratio of methanol and formate to sugar of from50:1 to 5:1. In certain embodiments, the sugar (e.g., glucose) isprovided at a molar concentration ratio of methanol and formate to sugarof 100:1. In one embodiment, the sugar (e.g., glucose) is provided at amolar concentration ratio of methanol and formate to sugar of 90:1. Inone embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 80:1. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 70:1. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 60:1. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 50:1. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 40:1. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 30:1. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 20:1. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 10:1. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 5:1. In oneembodiment. the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 2:1. In oneembodiment, the sugar (e.g. glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:1. In certainembodiments, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:100. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:90. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:80. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:70. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:60. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:50. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:40. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:30. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:20. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:10. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:5. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:2. In certainembodiments of the ratios provided above, the sugar is asugar-containing biomass.

Given the teachings and guidance provided herein, those skilled in theart will understand that a non-naturally occurring microbial organismcan be produced that secretes the biosynthesized compounds when grown ona carbon source such as a carbohydrate. Such compounds include, forexample, 1,2-propanediol, n-propanol, 1,3-propanediol or glycerol andany of the intermediate metabolites in the 1,2-propanediol, n-propanol,1,3-propanediol or glycerol pathway. All that is required is to engineerin one or more of the required enzyme or protein activities to achievebiosynthesis of the desired compound or intermediate including, forexample, inclusion of some or all of the 1,2-propanediol, n-propanol,1,3-propanediol or glycerol biosynthetic pathways. Accordingly, providedherein is a non-naturally occurring microbial organism that producesand/or secretes 1,2-propanediol, n-propanol, 1,3-propanediol or glycerolwhen grown on a carbohydrate or other carbon source and produces and/orsecretes any of the intermediate metabolites shown in the1,2-propanediol, n-propanol, 1,3-propanediol or glycerol pathway whengrown on a carbohydrate or other carbon source. The 1,2-propanediol-,n-propanol-, 1,3-propanediol- or glycerol-producing microbial organismsprovided herein can initiate synthesis from an intermediate. The sameholds true for intermediates in the formaldehyde assimilation andmethanol metabolic, pathways.

The non-naturally occurring microbial organisms provided herein areconstructed using methods well known in the art as exemplified herein toexogenously express at least one nucleic acid encoding a1,2-propanediol, n-propanol, 1,3-propanediol or glycerol biosyntheticpathway and/or methanol metabolic pathway enzyme or protein insufficient amounts to produce 1,2-propanediol, n-propanol,1,3-propanediol or glycerol. It is understood that the microbialorganisms are cultured under conditions sufficient to produce1,2-propanediol, n-propanol, 1,3-propanediol or glycerol. Following theteachings and guidance provided herein, the non-naturally occurringmicrobial organisms can achieve biosynthesis of 1,2-propanediol,n-propanol, 1,3-propanediol or glycerol, resulting in intracellularconcentrations between about 0.1-500 mM or more. Generally, theintracellular concentration of 1,2-propanediol, n-propanol,1,3-propanediol or glycerol is between about 3-150 mM, particularlybetween about 5-125 mM and more particularly between about 8-100 mM,including about 10 mM, 20 mM, 50 mM, 80 mM, or more. Intracellularconcentrations between and above each of these exemplary ranges also canbe achieved from the non-naturally occurring microbial organismsprovided herein.

In some embodiments, culture conditions include anaerobic orsubstantially anaerobic growth or maintenance conditions. Exemplaryanaerobic conditions have been described previously and are well knownin the art. Exemplary anaerobic conditions for fermentation processesare described herein and are described, for example, in U.S. Publ. No.2009/0047719. Any of these conditions can be employed with thenon-naturally occurring microbial organisms as well as other anaerobicconditions well known in the art. Under such anaerobic or substantiallyanaerobic conditions, the 1,2-propanediol, n-propanol, 1,3-propanediolor glycerol producers can synthesize 1,2-propanediol, n-propanol,1,3-propanediol or glycerol at intracellular concentrations of 5-100 mMor more as well as all other concentrations exemplified herein. It isunderstood that, even though the above description refers tointracellular concentrations, 1,2-propanediol, n-propanol,1,3-propanediol or glycerol can produce 1,2-propanediol, n-propanol,1,3-propanediol or glycerol intracellularly and/or secrete the productinto the culture medium.

Exemplary fermentation processes include, but are not limited to,fed-batch fermentation and batch separation; fed-batch fermentation andcontinuous separation; and continuous fermentation and continuousseparation. In an exemplary batch fermentation protocol, the productionorganism is grown in a suitably sized bioreactor sparged with anappropriate gas. Under anaerobic conditions, the culture is sparged withan inert gas or combination of gases, for example, nitrogen, N2/CO2mixture, argon, helium, and the like. As the cells grow and utilize thecarbon source, additional carbon source(s) and/or other nutrients arefed into the bioreactor at a rate approximately balancing consumption ofthe carbon source and/or nutrients. The temperature of the bioreactor ismaintained at a desired temperature, generally in the range of 22-37degrees C., but the temperature can be maintained at a higher or lowertemperature depending on the growth characteristics of the productionorganism and/or desired conditions for the fermentation process. Growthcontinues for a desired period of time to achieve desiredcharacteristics of the culture in the fermenter, for example, celldensity, product concentration, and the like. In a batch fermentationprocess, the time period for the fermentation is generally in the rangeof several hours to several days, for example, 8 to 24 hours, or 1, 2,3, 4 or 5 days, or up to a week, depending on the desired cultureconditions. The pH can be controlled or not, as desired, in which case aculture in which pH is not controlled will typically decrease to pH 3-6by the end of the run. Upon completion of the cultivation period, thefermenter contents can be passed through a cell separation unit, forexample, a centrifuge, filtration unit, and the like, to remove cellsand cell debris. In the case where the desired product is expressedintracellularly, the cells can be lysed or disrupted enzymatically orchemically prior to or after separation of cells from the fermentationbroth, as desired, in order to release additional product. Thefermentation broth can be transferred to a product separations unit.Isolation of product occurs by standard separations procedures employedin the art to separate a desired product from dilute aqueous solutions.Such methods include, but are not limited to, liquid-liquid extractionusing a water immiscible organic solvent (e.g., toluene or othersuitable solvents, including but not limited to diethyl ether, ethylacetate, tetrahydrofuran (THF), methylene chloride, chloroform, benzene,pentane, hexane, heptane, petroleum ether, methyl tertiary butyl ether(MTBE), dioxane, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), andthe like) to provide an organic solution of the product, if appropriate,standard distillation methods, and the like, depending on the chemicalcharacteristics of the product of the fermentation process.

In an exemplary fully continuous fermentation protocol, the productionorganism is generally first grown up in batch mode in order to achieve adesired cell density. When the carbon source and/or other nutrients areexhausted, feed medium of the same composition is supplied continuouslyat a desired rate, and fermentation liquid is withdrawn at the samerate. Under such conditions, the product concentration in the bioreactorgenerally remains constant, as well as the cell density. The temperatureof the fermenter is maintained at a desired temperature, as discussedabove. During the continuous fermentation phase, it is generallydesirable to maintain a suitable pH range for optimized production. ThepH can be monitored and maintained using routine methods, including theaddition of suitable acids or bases to maintain a desired pH range. Thebioreactor is operated continuously for extended periods of time,generally at least one week to several weeks and up to one month, orlonger, as appropriate and desired. The fermentation liquid and/orculture is monitored periodically, including sampling up to every day,as desired, to assure consistency of product concentration and/or celldensity. In continuous mode, fermenter contents are constantly removedas new feed medium is supplied. The exit stream, containing cells,medium, and product, are generally subjected to a continuous productseparations procedure, with or without removing cells and cell debris,as desired. Continuous separations methods employed in the art can beused to separate the product from dilute aqueous solutions, includingbut not limited to continuous liquid-liquid extraction using a waterimmiscible organic solvent (e.g., toluene or other suitable solvents,including but not limited to diethyl ether, ethyl acetate,tetrahydrofuran (THF), methylene chloride, chloroform, benzene, pentane,hexane, heptane, petroleum ether, methyl tertiary butyl ether (MTBE),dioxane, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), and thelike), standard continuous distillation methods, and the like, or othermethods well known in the art.

In addition to the culturing and fermentation conditions disclosedherein, growth condition for achieving biosynthesis of 1,2-propanediol,n-propanol, 1,3-propanediol or glycerol can include the addition of anosmoprotectant to the culturing conditions. In certain embodiments, thenon-naturally occurring microbial organisms provided herein can besustained, cultured or fermented as described herein in the presence ofan osmoprotectant. Briefly, an osmoprotectant refers to a compound thatacts as an osmolyte and helps a microbial organism as described hereinsurvive osmotic stress. Osmoprotectants include, but are not limited to,betaines, amino acids, and the sugar trehalose. Non-limiting examples ofsuch are glycine betaine, praline betaine, dimethylthetin,dimethylslfonioproprionate, 3-dimethylsulfonio-2-methylproprionate,pipecolic acid, dimethylsulfonioacetate, choline, L-carnitine andectoine. In one aspect, the osmoprotectant is glycine betaine. It isunderstood to one of ordinary skill in the art that the amount and typeof osmoprotectant suitable for protecting a microbial organism describedherein from osmotic stress will depend on the microbial organism used.The amount of osmoprotectant in the culturing conditions can be, forexample, no more than about 0.1 mM, no more than about 0.5 mM, no morethan about 1.0 mM, no more than about 1.5 mM, no more than about 2.0 mM,no more than about 2.5 mM, no more than about 3.0 mM, no more than about5.0 mM, no more than about 7.0 mM, no more than about 10 mM, no morethan about 50 mM, no more than about 100 mM or no more than about 500mM.

The culture conditions can include, for example, liquid cultureprocedures as well as fermentation and other large scale cultureprocedures. As described herein, particularly useful yields of thebiosynthetic products of the invention can be obtained under anaerobicor substantially anaerobic culture conditions.

As described herein, one exemplary growth condition for achievingbiosynthesis of 1,2-propanediol, n-propanol, 1,3-propanediol orglycerol, as well as other pathway intermediates, includes anaerobicculture or fermentation conditions. In certain embodiments, thenon-naturally occurring microbial organisms provided can be sustained,cultured or fermented under anaerobic or substantially anaerobicconditions. Briefly, anaerobic conditions refer to an environment devoidof oxygen. Substantially anaerobic conditions include, for example, aculture, batch fermentation or continuous fermentation such that thedissolved oxygen concentration in the medium remains between 0 and 10%of saturation. Substantially anaerobic conditions also includes growingor resting cells in liquid medium or on solid agar inside a sealedchamber maintained with an atmosphere of less than 1% oxygen. Thepercent of oxygen can be maintained by, for example, sparging theculture with an N₂/CO₂ mixture or other suitable non-oxygen gas orgases.

The culture conditions described herein can be scaled up and growncontinuously for manufacturing of 1,2-propanediol, n-propanol,1,3-propanediol or glycerol. Exemplary growth procedures include, forexample, fed-batch fermentation and batch separation; fed-batchfermentation and continuous separation, or continuous fermentation andcontinuous separation. All of these processes are well known in the art.Fermentation procedures are particularly useful for the biosyntheticproduction of commercial quantities of 1,2-propanediol, n-propanol,1,3-propanediol or glycerol. Generally, and as with non-continuousculture procedures, the continuous and/or near-continuous production of1,2-propanediol, n-propanol, 1,3-propanediol or glycerol will includeculturing a non-naturally occurring 1,2-propanediol, n-propanol,1,3-propanediol or glycerol producing organism of the invention insufficient nutrients and medium to sustain and/or nearly sustain growthin an exponential phase. Continuous culture under such conditions can beincluded, for example, growth for 1 day, 2, 3, 4, 5, 6 or 7 days ormore. Additionally, continuous culture can include longer time periodsof 1 week, 2, 3, 4 or 5 or more weeks and up to several months.Alternatively, organisms provided can be cultured for hours, if suitablefor a particular application. It is to be understood that the continuousand/or near-continuous culture conditions also can include all timeintervals in between these exemplary periods. It is further understoodthat the time of culturing the microbial organism of the invention isfor a sufficient period of time to produce a sufficient amount ofproduct for a desired purpose.

Fermentation procedures are well known in the art. Briefly, fermentationfor the biosynthetic production of 1,2-propanediol, n-propanol,1,3-propanediol or glycerol can be utilized in, for example, fed-batchfermentation and batch separation; fed-batch fermentation and continuousseparation, or continuous fermentation and continuous separation.Examples of batch and continuous fermentation procedures are well knownin the art.

In addition to the above fermentation procedures using the1,2-propanediol, n-propanol, 1,3-propanediol or glycerol producers forcontinuous production of substantial quantities of 1,2-propanediol,n-propanol, 1,3-propanediol or glycerol, the 1,2-propanediol,n-propanol, 1,3-propanediol or glycerol producers also can be, forexample, simultaneously subjected to chemical synthesis procedures toconvert the product to other compounds or the product can be separatedfrom the fermentation culture and sequentially subjected to chemicalconversion to convert the product to other compounds, if desired.

To generate better producers, metabolic modeling can be utilized tooptimize growth conditions. Modeling can also be used to design geneknockouts that additionally optimize utilization of the pathway (see,for example, U.S. Publ. Nos. 2002/0012939, 2003/0224363, 2004/0029149,2004/0072723, 2003/0059792, 2002/0168654 and 2004/0009466, and U.S. Pat.No. 7,127,379). Modeling analysis allows reliable predictions of theeffects on cell growth of shifting the metabolism towards more efficientproduction of 1,2-propanediol, n-propanol, 1,3-propanediol or glycerol.

One computational method for identifying and designing metabolicalterations favoring biosynthesis of a desired product is the OptKnockcomputational framework (Burgard et al., Biotechnol. Bioeng. 84:647-657(2003)). OptKnock is a metabolic modeling and simulation program thatsuggests gene deletion or disruption strategies that result ingenetically stable microorganisms which overproduce the target product.Specifically, the framework examines the complete metabolic and/orbiochemical network of a microorganism in order to suggest geneticmanipulations that force the desired biochemical to become an obligatorybyproduct of cell growth. By coupling biochemical production with cellgrowth through strategically placed gene deletions or other functionalgene disruption, the growth selection pressures imposed on theengineered strains after long periods of time in a bioreactor lead toimprovements in performance as a result of the compulsory growth-coupledbiochemical production. Lastly, when gene deletions are constructedthere is a negligible possibility of the designed strains reverting totheir wild-type states because the genes selected by OptKnock are to becompletely removed from the genome. Therefore, this computationalmethodology can be used to either identify alternative pathways thatlead to biosynthesis of a desired product or used in connection with thenon-naturally occurring microbial organisms for further optimization ofbiosynthesis of a desired product.

Briefly, OptKnock is a term used herein to refer to a computationalmethod and system for modeling cellular metabolism. The OptKnock programrelates to a framework of models and methods that incorporate particularconstraints into flux balance analysis (FBA) models. These constraintsinclude, for example, qualitative kinetic information, qualitativeregulatory information, and/or DNA microarray experimental data.OptKnock also computes solutions to various metabolic problems by, forexample, tightening the flux boundaries derived through flux balancemodels and subsequently probing the performance limits of metabolicnetworks in the presence of gene additions or deletions. OptKnockcomputational framework allows the construction of model formulationsthat allow an effective query of the performance limits of metabolicnetworks and provides methods for solving the resulting mixed-integerlinear programming problems. The metabolic modeling and simulationmethods referred to herein as OptKnock are described in, for example,U.S. Publ. No. 2002/0168654, International Patent Application No.PCT/US02/00660, and U.S. Publ. No. 2009/0047719.

Another computational method for identifying and designing metabolicalterations favoring biosynthetic production of a product is a metabolicmodeling and simulation system termed SimPheny®. This computationalmethod and system is described in, for example, U.S. Publ. No.2003/0233218, and International Patent Application No. PCT/US03/18838.SimPheny® is a computational system that can be used to produce anetwork model in silico and to simulate the flux of mass, energy orcharge through the chemical reactions of a biological system to define asolution space that contains any and all possible functionalities of thechemical reactions in the system, thereby determining a range of allowedactivities for the biological system. This approach is referred to asconstraints-based modeling because the solution space is defined byconstraints such as the known stoichiometry of the included reactions aswell as reaction thermodynamic and capacity constraints associated withmaximum fluxes through reactions. The space defined by these constraintscan be interrogated to determine the phenotypic capabilities andbehavior of the biological system or of its biochemical components.

These computational approaches are consistent with biological realitiesbecause biological systems are flexible and can reach the same result inmany different ways. Biological systems are designed throughevolutionary mechanisms that have been restricted by fundamentalconstraints that all living systems must face. Therefore,constraints-based modeling strategy embraces these general realities.Further, the ability to continuously impose further restrictions on anetwork model via the tightening of constraints results in a reductionin the size of the solution space, thereby enhancing the precision withwhich physiological performance or phenotype can be predicted.

Given the teachings and guidance provided herein, those skilled in theart will be able to apply various computational frameworks for metabolicmodeling and simulation to design and implement biosynthesis of adesired compound in host microbial organisms. Such metabolic modelingand simulation methods include, for example, the computational systemsexemplified above as SimPheny® and OptKnock. For illustration of theinvention, some methods are described herein with reference to theOptKnock computation framework for modeling and simulation. Thoseskilled in the art will know how to apply the identification, design andimplementation of the metabolic alterations using OptKnock to any ofsuch other metabolic modeling and simulation computational frameworksand methods well known in the art.

The methods described above will provide one set of metabolic reactionsto disrupt. Elimination of each reaction within the set or metabolicmodification can result in a desired product as an obligatory productduring the growth phase of the organism. Because the reactions areknown, a solution to the bilevel OptKnock problem also will provide theassociated gene or genes encoding one or more enzymes that catalyze eachreaction within the set of reactions. Identification of a set ofreactions and their corresponding genes encoding the enzymesparticipating in each reaction is generally an automated process,accomplished through correlation of the reactions with a reactiondatabase having a relationship between enzymes and encoding genes.

Once identified, the set of reactions that are to be disrupted in orderto achieve production of a desired product are implemented in the targetcell or organism by functional disruption of at least one gene encodingeach metabolic reaction within the set. One particularly useful means toachieve functional disruption of the reaction set is by deletion of eachencoding gene. However, in some instances, it can be beneficial todisrupt the reaction by other genetic aberrations including, forexample, mutation, deletion of regulatory regions such as promoters orcis binding sites for regulatory factors, or by truncation of the codingsequence at any of a number of locations. These latter aberrations,resulting in less than total deletion of the gene set can be useful, forexample, when rapid assessments of the coupling of a product are desiredor when genetic reversion is less likely to occur.

To identify additional productive solutions to the above describedbilevel OptKnock problem which lead to further sets of reactions todisrupt or metabolic modifications that can result in the biosynthesis,including growth-coupled biosynthesis of a desired product, anoptimization method, termed integer cuts, can be implemented. Thismethod proceeds by iteratively solving the OptKnock problem exemplifiedabove with the incorporation of an additional constraint referred to asan integer cut at each iteration. Integer cut constraints effectivelyprevent the solution procedure from choosing the exact same set ofreactions identified in any previous iteration that obligatorily couplesproduct biosynthesis to growth. For example, if a previously identifiedgrowth-coupled metabolic modification specifies reactions 1, 2, and 3for disruption, then the following constraint prevents the samereactions from being simultaneously considered in subsequent solutions.The integer cut method is well known in the art and can be founddescribed in, for example, Burgard et al., Biotechnol. Prog. 17:791-797(2001). As with all methods described herein with reference to their usein combination with the OptKnock computational framework for metabolicmodeling and simulation, the integer cut method of reducing redundancyin iterative computational analysis also can be applied with othercomputational frameworks well known in the art including, for example,SimPheny®.

The methods exemplified herein allow the construction of cells andorganisms that biosynthetically produce a desired product, including theobligatory coupling of production of a target biochemical product togrowth of the cell or organism engineered to harbor the identifiedgenetic alterations. Therefore, the computational methods describedherein allow the identification and implementation of metabolicmodifications that are identified by an in silico method selected fromOptKnock or SimPheny®. The set of metabolic modifications can include,for example, addition of one or more biosynthetic pathway enzymes and/orfunctional disruption of one or more metabolic reactions including, forexample, disruption by gene deletion.

As discussed above, the OptKnock methodology was developed on thepremise that mutant microbial networks can be evolved towards theircomputationally predicted maximum-growth phenotypes when subjected tolong periods of growth selection. In other words, the approach leveragesan organism's ability to self-optimize under selective pressures. TheOptKnock framework allows for the exhaustive enumeration of genedeletion combinations that force a coupling between biochemicalproduction and cell growth based on network stoichiometry. Theidentification of optimal gene/reaction knockouts requires the solutionof a bilevel optimization problem that chooses the set of activereactions such that an optimal growth solution for the resulting networkoverproduces the biochemical of interest (Burgard et al., Biotechnol.Bioeng. 84:647-657 (2003)).

An in silico stoichiometric model of E. coli metabolism can be employedto identify essential genes for metabolic pathways as exemplifiedpreviously and described in, for example, U.S. Publ. Nos. 2002/0012939,2003/0224363, 2004/0029149, 2004/0072723, 2003/0059792, 2002/0168654 and2004/0009466, and in U.S. Pat. No. 7,127,379. As disclosed herein, theOptKnock mathematical framework can be applied to pinpoint genedeletions leading to the growth-coupled production of a desired product.Further, the solution of the bilevel OptKnock problem provides only oneset of deletions. To enumerate all meaningful solutions, that is, allsets of knockouts leading to growth-coupled production formation, anoptimization technique, termed integer cuts, can be implemented. Thisentails iteratively solving the OptKnock problem with the incorporationof an additional constraint referred to as an integer cut at eachiteration, as discussed above.

As disclosed herein, a nucleic acid encoding a desired activity of a1,2-propanediol, n-propanol, 1,3-propanediol or glycerol pathway,formaldehyde assimilation pathway, and/or methanol metabolic pathway canbe introduced into a host organism. In some cases, it can be desirableto modify an activity of a 1,2-propanediol, n-propanol, 1,3-propanediolor glycerol pathway, formaldehyde assimilation, or methanol metabolicpathway enzyme or protein to increase production of 1,2-propanediol,n-propanol, 1,3-propanediol or glycerol; formaldehyde, and/or reducingequivalents. For example, known mutations that increase the activity ofa protein or enzyme can be introduced into an encoding nucleic acidmolecule. Additionally, optimization methods can be applied to increasethe activity of an enzyme or protein and/or decrease an inhibitoryactivity, for example, decrease the activity of a negative regulator.

One such optimization method is directed evolution. Directed evolutionis a powerful approach that involves the introduction of mutationstargeted to a specific gene in order to improve and/or alter theproperties of an enzyme. Improved and/or altered enzymes can beidentified through the development and implementation of sensitivehigh-throughput screening assays that allow the automated screening ofmany enzyme variants (for example, >10⁴). Iterative rounds ofmutagenesis and screening typically are performed to afford an enzymewith optimized properties. Computational algorithms that can help toidentify areas of the gene for mutagenesis also have been developed andcan significantly reduce the number of enzyme variants that need to begenerated and screened. Numerous directed evolution technologies havebeen developed (for reviews, see Hibbert et al., Biomol. Eng. 22:11-19(2005); Huisman and Lalonde, In Biocatalysis in the pharmaceutical andbiotechnology industries pgs. 717-742 (2007), Patel (ed.), CRC Press;Otten and Quax. Biomol. Eng. 22:1-9 (2005); and Sen et al., ApplBiochem. Biotechnol 143:212-223 (2007)) to be effective at creatingdiverse variant libraries, and these methods have been successfullyapplied to the improvement of a wide range of properties across manyenzyme classes. Enzyme characteristics that have been improved and/oraltered by directed evolution technologies include, for example:selectivity/specificity, for conversion of non-natural substrates;temperature stability, for robust high temperature processing; pHstability, for bioprocessing under lower or higher pH conditions;substrate or product tolerance, so that high product titers can beachieved; binding (K_(m)), including broadening substrate binding toinclude non-natural substrates; inhibition (K_(i)), to remove inhibitionby products, substrates, or key intermediates; activity (kcat), toincreases enzymatic reaction rates to achieve desired flux; expressionlevels, to increase protein yields and overall pathway flux; oxygenstability, for operation of air sensitive enzymes under aerobicconditions; and anaerobic activity, for operation of an aerobic enzymein the absence of oxygen.

A number of exemplary methods have been developed for the mutagenesisand diversification of genes to target desired properties of specificenzymes. Such methods are well known to those skilled in the art. Any ofthese can be used to alter and/or optimize the activity of a1,2-propanediol, n-propanol, 1,3-propanediol or glycerol pathway and/ora methanol metabolic pathway enzyme or protein. Such methods include,but are not limited to EpPCR, which introduces random point mutations byreducing the fidelity of DNA polymerase in PCR reactions (Pritchard etal., J. Theor. Biol. 234:497-509 (2005)); Error-prone Rolling CircleAmplification (epRCA), which is similar to epPCR except a whole circularplasmid is used as the template and random 6-mers with exonucleaseresistant thiophosphate linkages on the last 2 nucleotides are used toamplify the plasmid followed by transformation into cells in which theplasmid is re-circularized at tandem repeats (Fujii et al., NucleicAcids Res. 32:e145 (2004); and Fujii et al., Nat. Protocols 1:2493-2497(2006)); DNA or Family Shuffling, which typically involves digestion oftwo or more variant genes with nucleases such as Dnase I or EndoV togenerate a pool of random fragments that are reassembled by cycles ofannealing and extension in the presence of DNA polymerase to create alibrary of chimeric genes (Stemmer, Proc. Natl. Acad. Sci. U.S.A.91:10747-10751 (1994); and Stemmer, Nature 370:389-391 (1994));Staggered Extension (StEP), which entails template priming followed byrepeated cycles of 2 step PCR with denaturation and very short durationof annealing/extension (as short as 5 sec) (Zhao et al., Nat.Biotechnol. 16:258-261 (1998)); Random Priming Recombination (RPR), inwhich random sequence primers are used to generate many short DNAfragments complementary to different segments of the template (Shao etal., Nucleic Acids Res. 26:681-683 (1998)).

Additional methods include Heteroduplex Recombination, in whichlinearized plasmid DNA is used to form heteroduplexes that are repairedby mismatch repair (Volkov et al, Nucleic Acids Res. 27:e18 (1999); andVolkov et al., Methods Enzymol. 328:456-463 (2000)); RandomChimeragenesis on Transient Templates (RACHITT), which employs Dnase Ifragmentation and size fractionation of single stranded DNA (ssDNA)(Coco et al., Nat. Biotechnol. 19:354-359 (2001)); Recombined Extensionon Truncated templates (RETT), which entails template switching ofunidirectionally growing strands from primers in the presence ofunidirectional ssDNA fragments used as a pool of templates (Lee et al.,J. Molec. Catalysis 26:119-129 (2003)); Degenerate Oligonucleotide GeneShuffling (DOGS), in which degenerate primers are used to controlrecombination between molecules; (Bergquist and Gibbs, Methods Mol.Biol. 352:191-204 (2007); Bergquist et al., Biomol. Eng. 22:63-72(2005); Gibbs et al., Gene 271:13-20 (2001)); Incremental Truncation forthe Creation of Hybrid Enzymes (ITCHY), which creates a combinatoriallibrary with 1 base pair deletions of a gene or gene fragment ofinterest (Ostermeier et al., Proc. Natl. Acad. Sci. U.S.A. 96:3562-3567(1999); and Ostermeier et al., Nat. Biotechnol. 17:1205-1209 (1999));Thio-Incremental Truncation for the Creation of Hybrid Enzymes(THIO-ITCHY), which is similar to ITCHY except that phosphothioate dNTPsare used to generate truncations (Lutz et al., Nucleic Acids Res. 29:E16(2001)); SCRATCHY, which combines two methods for recombining genes,ITCHY and DNA shuffling (Lutz et al., Proc. Natl. Acad. Sci. U.S.A.98:11248-11253 (2001)); Random Drift Mutagenesis (RNDM), in whichmutations made via epPCR are followed by screening/selection for thoseretaining usable activity (Bergquist et al., Biomol. Eng. 22:63-72(2005)); Sequence Saturation Mutagenesis (SeSaM), a random mutagenesismethod that generates a pool of random length fragments using randomincorporation of a phosphothioate nucleotide and cleavage, which is usedas a template to extend in the presence of “universal” bases such asinosine, and replication of an inosine-containing complement givesrandom base incorporation and, consequently, mutagenesis (Wong et al.,Biotechnol. J. 3:74-82 (2008); Wong et al., Nucleic Acids Res. 32:e26(2004); and Wong et al., Anal. Biochem. 341:187-189 (2005)); SyntheticShuffling, which uses overlapping oligonucleotides designed to encode“all genetic diversity in targets” and allows a very high diversity forthe shuffled progeny (Ness et al., Nat. Biotechnol. 20:1251-1255(2002)); Nucleotide Exchange and Excision Technology NexT, whichexploits a combination of dUTP incorporation followed by treatment withuracil DNA glycosylase and then piperidine to perform endpoint DNAfragmentation (Muller et al., Nucleic Acids Res. 33:e117 (2005)).

Further methods include Sequence Homology-Independent ProteinRecombination (SHIPREC), in which a linker is used to facilitate fusionbetween two distantly related or unrelated genes, and a range ofchimeras is generated between the two genes, resulting in libraries ofsingle-crossover hybrids (Sieber et al., Nat. Biotechnol. 19:456-460(2001)); Gene Site Saturation Mutagenesis™ (GSSMT™), in which thestarting materials include a supercoiled double stranded DNA (dsDNA)plasmid containing an insert and two primers which are degenerate at thedesired site of mutations (Kretz et al., Methods Enzymol. 388:3-11(2004)); Combinatorial Cassette Mutagenesis (CCM), which involves theuse of short oligonucleotide cassettes to replace limited regions with alarge number of possible amino acid sequence alterations (Reidhaar-Olsonet al. Methods Enzymol. 208:564-586 (1991); and Reidhaar-Olson et al.Science 241:53-57 (1988)); Combinatorial Multiple Cassette Mutagenesis(CMCM), which is essentially similar to CCM and uses epPCR at highmutation rate to identify hot spots and hot regions and then extensionby CMCM to cover a defined region of protein sequence space (Reetz etal., Angew. Chem. Int. Ed Engl. 40:3589-3591 (2001)); the MutatorStrains technique, in which conditional is mutator plasmids, utilizingthe mutD5 gene, which encodes a mutant subunit of DNA polymerase III, toallow increases of 20 to 4000-X in random and natural mutation frequencyduring selection and block accumulation of deleterious mutations whenselection is not required (Selifonova et al., Appl. Environ. Microbiol.67:3645-3649 (2001)); Low et al., J. Mol. Biol. 260:359-3680 (1996)).

Additional exemplary methods include Look-Through Mutagenesis (LTM),which is a multidimensional mutagenesis method that assesses andoptimizes combinatorial mutations of selected amino acids (Rajpal etal., Proc. Natl. Acad. Sci. U.S.A. 102:8466-8471 (2005)); GeneReassembly, which is a DNA shuffling method that can be applied tomultiple genes at one time or to create a large library of chimeras(multiple mutations) of a single gene (Tunable GeneReassembly™ (TGR™)Technology supplied by Verenium Corporation), in Silico Protein DesignAutomation (PDA), which is an optimization algorithm that anchors thestructurally defined protein backbone possessing a particular fold, andsearches sequence space for amino acid substitutions that can stabilizethe fold and overall protein energetics, and generally works mosteffectively on proteins with known three-dimensional structures (Hayeset al., Proc. Natl. Acad. Sci. U.S.A. 99:15926-15931 (2002)); andIterative Saturation Mutagenesis (ISM), which involves using knowledgeof structure/function to choose a likely site for enzyme improvement,performing saturation mutagenesis at chosen site using a mutagenesismethod such as Stratagene QuikChange (Stratagene; San Diego Calif.),screening/selecting for desired properties, and, using improvedclone(s), starting over at another site and continue repeating until adesired activity is achieved (Reetz et al., Nat. Protocols 2:891-903(2007); and Reetz et al., Angew. Chem. Int. Ed Engl. 45:7745-7751(2006)).

Any of the aforementioned methods for mutagenesis can be used alone orin any combination. Additionally, any one or combination of the directedevolution methods can be used in conjunction with adaptive evolutiontechniques, as described herein.

Throughout this application various publications have been referenced.The disclosures of these publications in their entireties, includingGenBank and GI number publications, are hereby incorporated by referencein this application in order to more fully describe the state of the artto which this invention pertains. Although the invention has beendescribed with reference to the examples provided above, it should beunderstood that various modifications can be made without departing fromthe spirit of the invention.

It is understood that modifications which do not substantially affectthe activity of the various embodiments of this invention are alsoincluded within the definition of the invention provided herein.Accordingly, the following examples are intended to illustrate but notlimit the present invention.

4. EXAMPLES 4.1 Example I—Production of Reducing Equivalents Via aMethanol Metabolic Pathway

Exemplary methanol metabolic pathways are provided in FIG. 1.

FIG. 1, Step A—Methanol Methyltransferase

A complex of 3-methyltransferase proteins, denoted MtaA, MtaB, and MtaC,perform the desired methanol methyltransferase activity (Sauer et al.,Eur. J. Biochem. 243:670-677 (1997); Naidu and Ragsdale, J. Bacteriol.183:3276-3281 (2001); Tallant and Krzycki, J. Biol. Chem. 276:4485-4493(2001); Tallant and Krzycki, J. Bacteriol. 179:6902-6911 (1997); Tallantand Krzycki, J. Bacteriol. 178:1295-1301 (1996); Ragsdale, S. W., Crit.Rev. Biochem. Mol. Biol. 39:165-195 (2004)).

MtaB is a zinc protein that can catalyze the transfer of a methyl groupfrom methanol to MtaC, a corrinoid protein. Exemplary genes encodingMtaB and MtaC can be found in methanogenic archaea such asMethanosarcina barkeri (Maeder et al., J. Bacteriol. 188:7922-7931(2006) and Methanosarcina acetivorans (Galagan et al., Genome Res.12:532-542 (2002), as well as the acetogen, Moorella thermoacetica (Daset al., Proteins 67:167-176 (2007). In general, the MtaB and MtaC genesare adjacent to one another on the chromosome as their activities aretightly interdependent. The protein sequences of various MtaB and MtaCencoding genes in M. barkeri, M. acetivorans, and M. thermoaceticum canbe identified by their following GenBank accession numbers.

Protein GenBank ID GI number Organism MtaB1 YP_304299 73668284Methanosarcina barkeri MtaC1 YP_304298 73668283 Methanosarcina barkeriMtaB2 YP_307082 73671067 Methanosarcina barkeri MtaC2 YP_307081 73671066Methanosarcina barkeri MtaB3 YP_304612 73668597 Methanosarcina barkeriMtaC3 YP_304611 73668596 Methanosarcina barkeri MtaB1 NP_615421 20089346Methanosarcina acetivorans MtaB1 NP_615422 20089347 Methanosarcinaacetivorans MtaB2 NP_619254 20093179 Methanosarcina acetivorans MtaC2NP_619253 20093178 Methanosarcina acetivorans MtaB3 NP_616549 20090474Methanosarcina acetivorans MtaC3 NP_616550 20090475 Methanosarcinaacetivorans MtaB YP_430066 83590057 Moorella thermoacetica MtaCYP_430065 83590056 Moorella thermoacetica MtaA YP_430064 83590056Moorella thermoacetica

The MtaB1 and MtaC1 genes, YP_304299 and YP_304298, from M. barkeri werecloned into E. coli and sequenced (Sauer et al., Eur. J. Biochem.243:670-677 (1997)). The crystal structure of this methanol-cobalaminmethyltransferase complex is also available (Hagemeier et al., Proc.Natl. Acad. Sci. U.S.A. 103:18917-18922 (2006)). The MtaB genes,YP_307082 and YP_304612, in M. barkeri were identified by sequencehomology to YP_304299. In general, homology searches are an effectivemeans of identifying methanol methyltransferases because MtaB encodinggenes show little or no similarity to methyltransferases that act onalternative substrates such as trimethylamine, dimethylamine,monomethylamine, or dimethylsulfide. The MtaC genes, YP_307081 andYP_304611 were identified based on their proximity to the MtaB genes andalso their homology to YP_304298. The three sets of MtaB and MtaC genesfrom M. acetivorans have been genetically, physiologically, andbiochemically characterized (Pritchett and Metcalf, Mol. Microhiol.56:1183-1194 (2005)). Mutant strains lacking two of the sets were ableto grow on methanol, whereas a strain lacking all three sets of MtaB andMtaC genes sets could not grow on methanol. This suggests that each setof genes plays a role in methanol utilization. The M. thermoacetica MtaBgene was identified based on homology to the methanogenic MtaB genes andalso by its adjacent chromosomal proximity to the methanol-inducedcorrinoid protein, MtaC, which has been crystallized (Zhou et al., ActaCrystallogr. Sect. F. Struct. Biol. Cyrst. Commun. 61:537-540 (2005) andfurther characterized by Northern hybridization and Western Blotting((Das et al., Proteins 67:167-176 (2007)).

MtaA is zinc protein that catalyzes the transfer of the methyl groupfrom MtaC to either Coenzyme M in methanogens or methyltetrahydrofolatein acetogens. MtaA can also utilize methylcobalamin as the methyl donor.Exemplary genes encoding MtaA can be found in methanogenic archaea suchas Methanosarcina barkeri (Maeder et al., J. Bacteriol. 188:7922-7931(2006) and Methanosarcina acetivorans (Galagan et al., Genome Res.12:532-542 (2002), as well as the acetogen, Moorella thermoacetica ((Daset al., Proteins 67:167-176 (2007)). In general, MtaA proteins thatcatalyze the transfer of the methyl group from CH₃-MtaC are difficult toidentify bioinformatically as they share similarity to other corrinoidprotein methyltransferases and are not oriented adjacent to the MtaB andMtaC genes on the chromosomes. Nevertheless, a number of MtaA encodinggenes have been characterized. The protein sequences of these genes inM. barkeri and M. acetivorans can be identified by the following GenBankaccession numbers.

Protein GenBank ID GI number Organism MtaA YP_304602 73668587Methanosarcina barkeri MtaA1 NP_619241 20093166 Methanosarcinaacetivorans MtaA2 NP_616548 20090473 Methanosarcina acetivorans

The MtaA gene, YP_304602, from M. barkeri was cloned, sequenced, andfunctionally overexpressed in E. coli (Harms and Thauer, Eur. J.Biochem. 235:653-659 (1996)). In M. acetivorans, MtaA1 is required forgrowth on methanol, whereas MtaA2 is dispensable even though methaneproduction from methanol is reduced in MtaA2 mutants (Bose et al., J.Bacteriol. 190:4017-4026 (2008)). There are multiple additional MtaAhomologs in M. barkeri and M. acetivorans that are as yetuncharacterized, but may also catalyze corrinoid proteinmethyltransferase activity.

Putative MtaA encoding genes in M. thermoacetica were identified bytheir sequence similarity to the characterized methanogenic MtaA genes.Specifically, three M. thermoacetica genes show high homology (>30%sequence identity) to YP_304602 from M. barkeri. Unlike methanogenicMtaA proteins that naturally catalyze the transfer of the methyl groupfrom CH₃-MtaC to Coenzyme M, an M. thermoacetica MtaA is likely totransfer the methyl group to methyltetrahydrofolate given the similarroles of methyltetrahydrofolate and Coenzyme M in methanogens andacetogens, respectively. The protein sequences of putative MtaA encodinggenes from M. thermoacetica can be identified by the following Gen Bankaccession numbers.

Protein GenBank ID GI number Organism MtaA YP_430937 83590928 Moorellathermoacetica MtaA YP_431175 83591166 Moorella thermoacetica MtaAYP_430935 83590926 Moorella thermoacetica MtaA YP_430064 83590056Moorella thermoaceticaFIG. 1, Step B—Methylenetetrahydrofolate Reductase

The conversion of methyl-THF to methylenetetrahydrofolate is catalyzedby methylenetetrahydrofolate reductase. In M. thermoacetica, this enzymeis oxygen-sensitive and contains an iron-sulfur cluster (Clark andLjungdahl, J. Biol. Chem. 259:10845-10849 (1984). This enzyme is encodedby metF in E. coli (Sheppard et al., J. Bacteriol. 181:718-725 (1999)and CHY_1233 in C. hydrogenoformans (Wu et al., PLoS Genet. 1:e65(2005). The M. thermoacetica genes, and its C. hydrogenoformanscounterpart, are located near the CODH/ACS gene cluster, separated byputative hydrogenase and heterodisulfide reductase genes. Someadditional gene candidates found bioinformatically are listed below. InAcetobacterium woodii metF is coupled to the Rnf complex through RnfC2(Poehlein et al, PLoS One. 7:e33439). Homologs of RnfC are found inother organisms by blast search. The Rnf complex is known to be areversible complex (Fuchs (2011) Annu. Rev. Microbiol. 65:631-658).

Protein GenBank ID GI number Organism Moth_1191 YP_430048.1 83590039Moorella thermoacetica Moth_1192 YP_430049.1 83590040 Moorellathermoacetica metF NP_418376.1 16131779 Escherichia coli CHY_1233YP_360071.1 78044792 Carboxydothermus hydrogenoformans CLJU_c37610YP_003781889.1 300856905 Clostridium ljungdahlii DSM 13528DesfrDRAFT_3717 ZP_07335241.1 303248996 Desulfovibrio fructosovorans JJCcarbDRAFT_2950 ZP_05392950.1 255526026 Clostridium carboxidivorans P7Ccel74_010100023124 ZP_07633513.1 307691067 Clostridium cellulovorans743B Cphy_3110 YP_001560205.1 160881237 Clostridium phytofermentans ISDgFIG. 1, Steps C and D—Methylenetetrahydrofolate Dehydrogenase,Methenyltetrahydrofolate Cyclohydrolase

In M. thermoacetica, E. coli, and C. hydrogenoformans,methenyltetrahydrofolate cyclohydrolase and methylenetetrahydrofolatedehydrogenase are carried out by the bi-functional gene products ofMoth_1516, folD, and CHY_1878, respectively (Pierce et al., Environ.Microbiol. 10:2550-2573 (2008); Wu et al., PLoS Genet. 1:e65 (2005);D'Ari and Rabinowitz, J. Biol. Chem. 266:23953-23958 (1991)). A homologexists in C. carboxidivorans P7. Several other organisms also encode forthis bifunctional protein as tabulated below.

Protein GenBank ID GI number Organism Moth_1516 YP_430368.1 83590359Moorella thermoacetica folD NP_415062.1 16128513 Escherichia coliCHY_1878 YP_360698.1 78044829 Carboxydothermus hydrogenoformansCcarbDRAFT_2948 ZP_05392948.1 255526024 Clostridium carboxidivorans P7folD ADK16789.1 300437022 Clostridium ljungdahlii DSM 13528 folD-2NP_951919.1 39995968 Geobacter sulfurreducens PCA folD YP_725874.1113867385 Ralstonia eutropha H16 folD NP_348702.1 15895353 Clostridiumacetobutylicum ATCC 824 folD YP_696506.1 110800457 Clostridiumperfringens MGA3_09460 EIJ83438.1 387591119 Bacillus methanolicus MGA3PB1_14689 ZP_10132349.1 387929672 Bacillus methanolicus PB1FIG. 1, Step E—Formyltetrahydrofolate Deformylase

This enzyme catalyzes the hydrolysis of 10-formyltetrahydrofolate(formyl-THF) to THF and formate. In E. coli, this enzyme is encoded bypurU and has been overproduced, purified, and characterized (Nagy, etal., J. Bacteriol. 3:1292-1298 (1995)). Homologs exist inCorynebacterium sp. U-96 (Suzuki, et al., Biosci. Biotechnol. Biochem.69(5):952-956 (2005)), Corynebacterium glutamicum ATCC 14067, Salmonellaenterica, and several additional organisms.

Protein GenBank ID GI number Organism purU AAC74314.1 1787483Escherichia coli K-12 MG1655 purU BAD97821.1 63002616 Corynebacteriumsp. U-96 purU EHE84645.1 354511740 Corynebacterium glutamicum ATCC 14067purU NP_460715.1 16765100 Salmonella enterica subsp. enterica serovarTyphimurium str. LT2FIG. 1, Step F—Formyltetrahydrofolate Synthetase

Formyltetrahydrofolate synthetase ligates formate to tetrahydrofolate atthe expense of one ATP. This reaction is catalyzed by the gene productof Moth_0109 in M. thermoacetica (O'brien et al., Experientia Suppl.26:249-262 (1976); Lovell et al., Arch. Microbiol. 149:280-285 (1988);Lovell et al., Biochemistry 29:5687-5694 (1990)), FHS in Clostridiumacidurici (Whitehead and Rabinowitz, J. Bacteriol. 167:203-209 (1986);Whitehead and Rabinowitz, J. Bacteriol. 170:3255-3261 (1988), andCHY_2385 in C. hydrogenoformans (Wu et al., PLoS Genet. 1:e65 (2005).Homologs exist in C. carboxidivorans P7. This enzyme is found in severalother organisms as listed below.

Protein GenBank ID GI number Organism Moth_0109 YP_428991.1 83588982Moorella thermoacetica CHY_2385 YP_361182.1 78045024 Carboxydothermushydrogenoformans FHS P13419.1 120562 Clostridium aciduriciCcarbDRAFT_1913 ZP_05391913.1 255524966 Clostridium carboxidivorans P7CcarbDRAFT_2946 ZP_05392946.1 255526022 Clostridium carboxidivorans P7Dhaf_0555 ACL18622.1 219536883 Desulfitobacterium hafniense FhsYP_001393842.1 153953077 Clostridium kluyveri DSM 555 Fhs YP_003781893.1300856909 Clostridium ljungdahlii DSM 13528 MGA3_08300 EIJ83208.1387590889 Bacillus methanolicus MGA3 PB1_13509 ZP_10132113.1 387929436Bacillus methanolicus PB1FIG. 1, Step G—Formate Hydrogen Lyase

A formate hydrogen lyase enzyme can be employed to convert formate tocarbon dioxide and hydrogen. An exemplary formate hydrogen lyase enzymecan be found in Escherichia coli. The E. coli formate hydrogen lyaseconsists of hydrogenase 3 and formate dehydrogenase-H (Maeda et al.,Appl Microbiol Biotechnol 77:879-890 (2007)). It is activated by thegene product of fhlA. (Maeda et al., Appl Microbiol Biotechnol77:879-890 (2007)). The addition of the trace elements, selenium, nickeland molybdenum, to a fermentation broth has been shown to enhanceformate hydrogen lyase activity (Soini et al., Microb. Cell Fact. 7:26(2008)). Various hydrogenase 3, formate dehydrogenase andtranscriptional activator genes are shown below.

Protein GenBank ID GI number Organism hycA NP_417205 16130632Escherichia coli K-12 MG1655 hycB NP_417204 16130631 Escherichia coliK-12 MG1655 hycC NP_417203 16130630 Escherichia coli K-12 MG1655 hycDNP_417202 16130629 Escherichia coli K-12 MG1655 hycE NP_417201 16130628Escherichia coli K-12 MG1655 hycF NP_417200 16130627 Escherichia coliK-12 MG1655 hycG NP_417199 16130626 Escherichia coli K-12 MG1655 hycHNP_417198 16130625 Escherichia coli K-12 MG1655 hycI NP_417197 16130624Escherichia coli K-12 MG1655 fdhF NP_418503 16131905 Escherichia coliK-12 MG1655 fhlA NP_417211 16130638 Escherichia coli K-12 MG1655

A formate hydrogen lyase enzyme also exists in the hyperthermophilicarchaeon, Thermococcus litoralis (Takacs et al., BMC. Microbiol 8:88(2008)).

Protein GenBank ID GI number Organism mhyC ABW05543 157954626Thermococcus litoralis mhyD ABW05544 157954627 Thermococcus litoralismhyE ABW05545 157954628 Thermococcus litoralis myhF ABW05546 157954629Thermococcus litoralis myhG ABW05547 157954630 Thermococcus litoralismyhH ABW05548 157954631 Thermococcus litoralis fdhA AAB94932 2746736Thermococcus litoralis fdhB AAB94931 157954625 Thermococcus litoralis

Additional formate hydrogen lyase systems have been found in Salmonellatyphimurium, Klebsiella pneumoniae, Rhodospirillum rubrum,Methanobacterium formicicum (Vardar-Schara et al., MicrobialBiotechnology 1:107-125 (2008)).

FIG. 1, Step H—Hydrogenase

Hydrogenase enzymes can convert hydrogen gas to protons and transferelectrons to acceptors such as ferredoxins, NAD+, or NADP+. Ralstoniaeutropha H16 uses hydrogen as an energy source with oxygen as a terminalelectron acceptor. Its membrane-bound uptake [NiFe]-hydrogenase is an“O2-tolerant” hydrogenase (Cracknell, et al. Proc Nat Acad Sci, 106(49)20681-20686 (2009)) that is periplasmically-oriented and connected tothe respiratory chain via a b-type cytochrome (Schink and Schlegel,Biochim. Biophys. Acta, 567, 315-324 (1979); Bernhard et al., Eur. J.Biochem. 248, 179-186 (1997)). R. eutropha also contains an O₂-tolerantsoluble hydrogenase encoded by the Hox operon which is cytoplasmic anddirectly reduces NAD+ at the expense of hydrogen (Schneider andSchlegel, Biochim. Biophys. Acta 452, 66-80 (1976); Burgdorf, J. Bact.187(9) 3122-3132 (2005)). Soluble hydrogenase enzymes are additionallypresent in several other organisms including Geobacter sulfurreducens(Coppi, Microbiology 151, 1239-1254 (2005)), Synechocystis str. PCC 6803(Germer, J. Biol. Chem., 284(52), 36462-36472 (2009)), and Thiocapsaroseopersicina (Rakhely, Appl. Environ. Microbiol. 70(2) 722-728(2004)). The Synechocystis enzyme is capable of generating NADPH fromhydrogen. Overexpression of both the Hox operon from Synechocystis str.PCC 6803 and the accessory genes encoded by the Hyp operon from Nostocsp. PCC 7120 led to increased hydrogenase activity compared toexpression of the Hox genes alone (Germer, J. Biol. Chem. 284(52),36462-36472 (2009)).

Protein GenBank ID GI Number Organism HoxF NP_942727.1 38637753Ralstonia eutropha H16 HoxU NP_942728.1 38637754 Ralstonia eutropha H16HoxY NP_942729.1 38637755 Ralstonia eutropha H16 HoxH NP_942730.138637756 Ralstonia eutropha H16 HoxW NP_942731.1 38637757 Ralstoniaeutropha H16 HoxI NP_942732.1 38637758 Ralstonia eutropha H16 HoxENP_953767.1 39997816 Geobacter sulfurreducens HoxF NP_953766.1 39997815Geobacter sulfurreducens HoxU NP_953765.1 39997814 Geobactersulfurreducens HoxY NP_953764.1 39997813 Geobacter sulfurreducens HoxHNP_953763.1 39997812 Geobacter sulfurreducens GSU2717 NP_953762.139997811 Geobacter sulfurreducens HoxE NP_441418.1 16330690Synechocystis str. PCC 6803 HoxF NP_441417.1 16330689 Synechocystis str.PCC 6803 Unknown NP_441416.1 16330688 Synechocystis str. PCC 6803function HoxU NP_441415.1 16330687 Synechocystis str. PCC 6803 HoxYNP_441414.1 16330686 Synechocystis str. PCC 6803 Unknown NP_441413.116330685 Synechocystis str. PCC 6803 function Unknown NP_441412.116330684 Synechocystis str. PCC 6803 function HoxH NP_441411.1 16330683Synechocystis str. PCC 6803 HypF NP_484737.1 17228189 Nostoc sp. PCC7120 HypC NP_484738.1 17228190 Nostoc sp. PCC 7120 HypD NP_484739.117228191 Nostoc sp. PCC 7120 Unknown NP_484740.1 17228192 Nostoc sp. PCC7120 function HypE NP_484741.1 17228193 Nostoc sp. PCC 7120 HypANP_484742.1 17228194 Nostoc sp. PCC 7120 HypB NP_484743.1 17228195Nostoc sp. PCC 7120 Hox1E AAP50519.1 37787351 Thiocapsa roseopersicinaHox1F AAP50520.1 37787352 Thiocapsa roseopersicina Hox1U AAP50521.137787353 Thiocapsa roseopersicina Hox1Y AAP50522.1 37787354 Thiocapsaroseopersicina Hox1H AAP50523.1 37787355 Thiocapsa roseopersicina

The genomes of E. coli and other enteric bacteria encode up to fourhydrogenase enzymes (Sawers, G., Antonie Van Leeuwenhoek 66:57-88(1994); Sawers et al., J Bacteriol. 164:1324-1331 (1985); Sawers andBoxer, Eur. J Biochem. 156:265-275 (1986); Sawers et al., J Bacteriol.168:398-404 (1986)). Given the multiplicity of enzyme activities E. colior another host organism can provide sufficient hydrogenase activity tosplit incoming molecular hydrogen and reduce the corresponding acceptor.Endogenous hydrogen-lyase enzymes of E. coli include hydrogenase 3, amembrane-bound enzyme complex using ferredoxin as an acceptor, andhydrogenase 4 that also uses a ferredoxin acceptor. Hydrogenase 3 and 4are encoded by the hyc and hyf gene clusters, respectively. Hydrogenaseactivity in E. coli is also dependent upon the expression of the hypgenes whose corresponding proteins are involved in the assembly of thehydrogenase complexes (Jacobi et al., Arch. Microbiol 158:444-451(1992); Rangarajan et al., J Bacteriol. 190:1447-1458 (2008)). The M.thermoacetica and Clostridium ljungdahli hydrogenases are suitable for ahost that lacks sufficient endogenous hydrogenase activity. M.thermoacetica and C. ljungdahli can grow with CO₂ as the exclusivecarbon source indicating that reducing equivalents are extracted from H₂to enable acetyl-CoA synthesis via the Wood-Ljungdahl pathway (Drake, H.L., J Bacteriol. 150:702-709 (1982); Drake and Daniel, Res Microbiol155:869-883 (2004); Kellum and Drake, J Bacteriol. 160:466-469 (1984)).M. thermoacetica has homologs to several hyp, hyc, and hyf genes from E.coli. These protein sequences encoded for by these genes are identifiedby the following GenBank accession numbers. In addition, several geneclusters encoding hydrogenase functionality are present in M.thermoacetica and C. ljungdahli (see for example US 2012/0003652).

Protein GenBank ID GI Number Organism HypA NP_417206 16130633Escherichia coli HypB NP_417207 16130634 Escherichia coli HypC NP_41720816130635 Escherichia coli HypD NP_417209 16130636 Escherichia coli HypENP_417210 226524740 Escherichia coli HypF NP_417192 16130619 Escherichiacoli HycA NP_417205 16130632 Escherichia coli HycB NP_417204 16130631Escherichia coli HycC NP_417203 16130630 Escherichia coli HycD NP_41720216130629 Escherichia coli HycE NP_417201 16130628 Escherichia coli HycFNP_417200 16130627 Escherichia coli HycG NP_417199 16130626 Escherichiacoli HycH NP_417198 16130625 Escherichia coli HycI NP_417197 16130624Escherichia coli HyfA NP_416976 90111444 Escherichia coli HyfB NP_41697716130407 Escherichia coli HyfC NP_416978 90111445 Escherichia coli HyfDNP_416979 16130409 Escherichia coli HyfE NP_416980 16130410 Escherichiacoli HyfF NP_416981 16130411 Escherichia coli HyfG NP_416982 16130412Escherichia coli HyfH NP_416983 16130413 Escherichia coli HyfI NP_41698416130414 Escherichia coli HyfJ NP_416985 90111446 Escherichia coli HyfRNP_416986 90111447 Escherichia coli

Proteins in M. thermoacetica whose genes are homologous to the E. colihydrogenase genes are shown below.

Protein GenBank ID GI Number Organism Moth_2175 YP_431007 83590998Moorella thermoacetica Moth_2176 YP_431008 83590999 Moorellathermoacetica Moth_2177 YP_431009 83591000 Moorella thermoaceticaMoth_2178 YP_431010 83591001 Moorella thermoacetica Moth_2179 YP_43101183591002 Moorella thermoacetica Moth_2180 YP_431012 83591003 Moorellathermoacetica Moth_2181 YP_431013 83591004 Moorella thermoaceticaMoth_2182 YP_431014 83591005 Moorella thermoacetica Moth_2183 YP_43101583591006 Moorella thermoacetica Moth_2184 YP_431016 83591007 Moorellathermoacetica Moth_2185 YP_431017 83591008 Moorella thermoaceticaMoth_2186 YP_431018 83591009 Moorella thermoacetica Moth_2187 YP_43101983591010 Moorella thermoacetica Moth_2188 YP_431020 83591011 Moorellathermoacetica Moth_2189 YP_431021 83591012 Moorella thermoaceticaMoth_2190 YP_431022 83591013 Moorella thermoacetica Moth_2191 YP_43102383591014 Moorella thermoacetica Moth_2192 YP_431024 83591015 Moorellathermoacetica Moth_0439 YP_429313 83589304 Moorella thermoaceticaMoth_0440 YP_429314 83589305 Moorella thermoacetica Moth_0441 YP_42931583589306 Moorella thermoacetica Moth_0442 YP_429316 83589307 Moorellathermoacetica Moth_0809 YP_429670 83589661 Moorella thermoaceticaMoth_0810 YP_429671 83589662 Moorella thermoacetica Moth_0811 YP_42967283589663 Moorella thermoacetica Moth_0812 YP_429673 83589664 Moorellathermoacetica Moth_0814 YP_429674 83589665 Moorella thermoaceticaMoth_0815 YP_429675 83589666 Moorella thermoacetica Moth_0816 YP_42967683589667 Moorella thermoacetica Moth_1193 YP_430050 83590041 Moorellathermoacetica Moth_1194 YP_430051 83590042 Moorella thermoaceticaMoth_1195 YP_430052 83590043 Moorella thermoacetica Moth_1196 YP_43005383590044 Moorella thermoacetica Moth_1717 YP_430562 83590553 Moorellathermoacetica Moth_1718 YP_430563 83590554 Moorella thermoaceticaMoth_1719 YP_430564 83590555 Moorella thermoacetica Moth_1883 YP_43072683590717 Moorella thermoacetica Moth_1884 YP_430727 83590718 Moorellathermoacetica Moth_1885 YP_430728 83590719 Moorella thermoaceticaMoth_1886 YP_430729 83590720 Moorella thermoacetica Moth_1887 YP_43073083590721 Moorella thermoacetica Moth_1888 YP_430731 83590722 Moorellathermoacetica Moth_1452 YP_430305 83590296 Moorella thermoaceticaMoth_1453 YP_430306 83590297 Moorella thermoacetica Moth_1454 YP_43030783590298 Moorella thermoacetica

Genes encoding hydrogenase enzymes from C. ljungdahli are shown below.

Protein GenBank ID GI Number Organism CLJU_c20290 ADK15091.1 300435324Clostridium ljungdahli CLJU_c07030 ADK13773.1 300434006 Clostridiumljungdahli CLJU_c07040 ADK13774.1 300434007 Clostridium ljungdahliCLJU_c07050 ADK13775.1 300434008 Clostridium jungdahli CLJU_c07060ADK13776.1 300434009 Clostridium ljungdahli CLJU_c07070 ADK13777.1300434010 Clostridium ljungdahli CLJU_c07080 ADK13778.1 300434011Clostridium ljungdahli CLJU_c14730 ADK14541.1 300434774 Clostridiumljungdahli CLJU_c14720 ADK14540.1 300434773 Clostridium ljungdahliCLJU_c14710 ADK14539.1 300434772 Clostridium ljungdahli CLJU_c14700ADK14538.1 300434771 Clostridium ljungdahli CLJU_c28670 ADK15915.1300436148 Clostridium ljungdahli CLJU_c28660 ADK15914.1 300436147Clostridium ljungdahli CLJU_c28650 ADK15913.1 300436146 Clostridiumljungdahli CLJU_c28640 ADK15912.1 300436145 Clostridium ljungdahli

In some cases, hydrogenase encoding genes are located adjacent to aCODH. In Rhodospirillum rubrum, the encoded CODH/hydrogenase proteinsform a membrane-bound enzyme complex that has been indicated to be asite where energy, in the form of a proton gradient, is generated fromthe conversion of CO and H₂O to CO₂ and H₂ (Fox et al., J Bacteriol.178:6200-6208 (1996)). The CODH-I of C. hydrogenoformans and itsadjacent genes have been proposed to catalyze a similar functional rolebased on their similarity to the R. rubrum CODH/hydrogenase gene cluster(Wu et al., PLoS Genet. 1:e65 (2005)). The C. hydrogenoformans CODH-Iwas also shown to exhibit intense CO oxidation and CO₂ reductionactivities when linked to an electrode (Parkin et al., J Am. Chem. Soc.129:10328-10329 (2007)).

Protein GenBank ID GI Number Organism CooL AAC45118 1515468Rhodospirillum rubrum CooX AAC45119 1515469 Rhodospirillum rubrum CooUAAC45120 1515470 Rhodospirillum rubrum CooH AAC45121 1498746Rhodospirillum rubrum CooF AAC45122 1498747 Rhodospirillum rubrum CODH(CooS) AAC45123 1498748 Rhodospirillum rubrum CooC AAC45124 1498749Rhodospirillum rubrum CooT AAC45125 1498750 Rhodospirillum rubrum CooJAAC45126 1498751 Rhodospirillum rubrum CODH-I (CooS-I) YP_36064478043418 Carboxydothermus hydrogenoformans CooF YP_360645 78044791Carboxydothermus hydrogenoformans HypA YP_360646 78044340Carboxydothermus hydrogenoformans CooH YP_360647 78043871Carboxydothermus hydrogenoformans CooU YP_360648 78044023Carboxydothermus hydrogenoformans CooX YP_360649 78043124Carboxydothermus hydrogenoformans CooL YP_360650 78043938Carboxydothermus hydrogenoformans CooK YP_360651 78044700Carboxydothermus hydrogenoformans CooM YP_360652 78043942Carboxydothermus hydrogenoformans CooC YP_360654.1 78043296Carboxydothermus hydrogenoformans CooA-1 YP_360655.1 78044021Carboxydothermus hydrogenoformans

Some hydrogenase and CODH enzymes transfer electrons to ferredoxins.Ferredoxins are small acidic proteins containing one or more iron-sulfurclusters that function as intracellular electron carriers with a lowreduction potential. Reduced ferredoxins donate electrons toFe-dependent enzymes such as ferredoxin-NADP⁺ oxidoreductase,pyruvate:ferredoxin oxidoreductase (PFOR) and 2-oxoglutarate:ferredoxinoxidoreductase (OFOR). The H. thermophilus gene fdx1 encodes a[4Fe-4S]-type ferredoxin that is required for the reversiblecarboxylation of 2-oxoglutarate and pyruvate by OFOR and PFOR,respectively (Yamamoto et al., Extremophiles 14:79-85 (2010)). Theferredoxin associated with the Sulfolobus solfataricus2-oxoacid:ferredoxin reductase is a monomeric dicluster [3Fe-4S][4Fe-4S]type ferredoxin (Park et al. 2006). While the gene associated with thisprotein has not been fully sequenced, the N-terminal domain shares 93%homology with the zfx ferredoxin from S. acidocaldarius. The E. coligenome encodes a soluble ferredoxin of unknown physiological function,fdx. Some evidence indicates that this protein can function iniron-sulfur cluster assembly (Takahashi and Nakamura, 1999). Additionalferredoxin proteins have been characterized in Helicobacter pylori(Mukhopadhyay et al. 2003) and Campylobacter jejuni (van Vliet et al.2001). A 2Fe-2S ferredoxin from Clostridium pasteurianum has been clonedand expressed in E. coli (Fujinaga and Meyer, Biochemical andBiophysical Research Communications, 192(3): (1993)). Acetogenicbacteria such as Moorella thermoacetica, Clostridium carboxidivorans P7.Clostridium ljungdahli and Rhodospirillum rubrum are predicted to encodeseveral ferredoxins, listed below.

Protein GenBank ID GI Number Organism fdx1 BAE02673.1 68163284Hydrogenobacter thermophilus M11214.1 AAA83524.1 144806 Clostridiumpasteurianum Zfx AAY79867.1 68566938 Sulfolobus acidocalarius FdxAAC75578.1 1788874 Escherichia coli hp_0277 AAD07340.1 2313367Helicobacter pylori fdxA CAL34484.1 112359698 Campylobacter jejuniMoth_0061 ABC18400.1 83571848 Moorella thermoacetica Moth_1200ABC19514.1 83572962 Moorella thermoacetica Moth_1888 ABC20188.1 83573636Moorella thermoacetica Moth_2112 ABC20404.1 83573852 Moorellathermoacetica Moth_1037 ABC19351.1 83572799 Moorella thermoaceticaCcarbDRAFT_4383 ZP_05394383.1 255527515 Clostridium carboxidivorans P7CcarbDRAFT_2958 ZP_05392958.1 255526034 Clostridium carboxidivorans P7CcarbDRAFT_2281 ZP_05392281.1 255525342 Clostridium carboxidivorans P7CcarbDRAFT_5296 ZP_05395295.1 255528511 Clostridium carboxidivorans P7CcarbDRAFT_1615 ZP_05391615.1 255524662 Clostridium carboxidivorans P7CcarbDRAFT_1304 ZP_05391304.1 255524347 Clostridium carboxidivorans P7cooF AAG29808.1 11095245 Carboxydothermus hydrogenoformans fdxNCAA35699.1 46143 Rhodobacter capsulatus Rru_A2264 ABC23064.1 83576513Rhodospirillum rubrum Rru_A1916 ABC22716.1 83576165 Rhodospirillumrubrum Rru_A2026 ABC22826.1 83576275 Rhodospirillum rubrum cooFAAC45122.1 1498747 Rhodospirillum rubrum fdxN AAA26460.1 152605Rhodospirillum rubrum Alvin_2884 ADC63789.1 288897953 Allochromatiumvinosum DSM 180 Fdx YP_002801146.1 226946073 Azotobacter vinelandii DJCKL_3790 YP_001397146.1 153956381 Clostridium kluyveri DSM 555 fer1NP_949965.1 39937689 Rhodopseudomonas palustris CGA009 Fdx CAA12251.13724172 Thauera aromatica CHY_2405 YP_361202.1 78044690 Carboxydothermushydrogenoformans Fer YP_359966.1 78045103 Carboxydothermushydrogenoformans Fer AAC83945.1 1146198 Bacillus subtilis fdx1NP_249053.1 15595559 Pseudomonas aeruginosa PA01 yfhL AP_003148.189109368 Escherichia coli K-12 CLJU_c00930 ADK13195.1 300433428Clostridium ljungdahli CLJU_c00010 ADK13115.1 300433348 Clostridiumljungdahli CLJU_c01820 ADK13272.1 300433505 Clostridium ljungdahliCLJU_c17980 ADK14861.1 300435094 Clostridium ljungdahli CLJU_c17970ADK14860.1 300435093 Clostridium ljungdahli CLJU_c22510 ADK15311.1300435544 Clostridium ljungdahli CLJU_c26680 ADK15726.1 300435959Clostridium ljungdahli CLJU_c29400 ADK15988.1 300436221 Clostridiumljungdahli

Ferredoxin oxidoreductase enzymes transfer electrons from ferredoxins orflavodoxins to NAD(P)H. Two enzymes catalyzing the reversible transferof electrons from reduced ferredoxins to NAD(P)+ are ferredoxin:NAD+oxidoreductase (EC 1.18.1.3) and ferredoxin:NADP+ oxidoreductase (FNR,EC 1.18.11). Ferredoxin:NADP+ oxidoreductase (FNR, EC 1.18.1.2) has anoncovalently bound FAD cofactor that facilitates the reversibletransfer of electrons from NADPH to low-potential acceptors such asferredoxins or flavodoxins (Blaschkowski et al., Eur. J. Biochem.123:563-569 (1982); Fujii et al., 1977). The Helicobacter pylori FNR,encoded by HP1164 (fqrB), is coupled to the activity ofpyruvate:ferredoxin oxidoreductase (PFOR) resulting in thepyruvate-dependent production of NADPH (St et al. 2007). An analogousenzyme is found in Campylobacter jejuni (St Maurice et al., J.Bacteriol. 189:4764-4773 (2007)). A ferredoxin:NADP+ oxidoreductaseenzyme is encoded in the E. coli genome by fpr (Bianchi et al. 1993).Ferredoxin:NAD+ oxidoreductase utilizes reduced ferredoxin to generateNADH from NAD⁺. In several organising, including E. coli, this enzyme isa component of multifunctional dioxygenase enzyme complexes. Theferredoxin:NAD+ oxidoreductase of E. coli, encoded by hcaD, is acomponent of the 3-phenylproppionate dioxygenase system involved ininvolved in aromatic acid utilization (Diaz et al. 1998).NADH:ferredoxin reductase activity was detected in cell extracts ofHydrogenobacter thermophilus, although a gene with this activity has notyet been indicated (Yoon et al. 2006). Additional ferredoxin:NAD(P)+oxidoreductases have been annotated in Clostridium carboxydivorans P7.The NADH-dependent reduced ferredoxin: NADP oxidoreductase of C.kluyveri, encoded by nfnAB, catalyzes the concomitant reduction offerredoxin and NAD+ with two equivalents of NADPH (Wang et al, JBacteriol 192: 5115-5123 (2010)). Finally, the energy-conservingmembrane-associated Rnf-type proteins (Seedorf et al, PNAS 105:2128-2133(2008); and Herrmann, J. Bacteriol 190:784-791 (2008)) provide a meansto generate NADH or NADPH from reduced ferredoxin.

Protein GenBank ID GI Number Organism fqrB NP_207955.1 15645778Helicobacter pylori fqrB YP_001482096.1 157414840 Campylobacter jejuniRPA3954 CAE29395.1 39650872 Rhodopseudomonas palustris Fpr BAH29712.1225320633 Hydrogenobacter thermophilus yumC NP_391091.2 255767736Bacillus subtilis Fpr P28861.4 399486 Escherichia coli hcaD AAC75595.11788892 Escherichia coli LOC100282643 NP_001149023.1 226497434 Zea maysNfnA YP_001393861.1 153953096 Clostridium kluyveri NfnB YP_001393862.1153953097 Clostridium kluyveri CcarbDRAFT_2639 ZP_05392639.1 255525707Clostridium carboxidivorans P7 CcarbDRAFT_2638 ZP_05392638.1 255525706Clostridium carboxidivorans P7 CcarbDRAFT_2636 ZP_05392636.1 255525704Clostridium carboxidivorans P7 CcarbDRAFT_5060 ZP_05395060.1 255528241Clostridium carboxidivorans P7 CcarbDRAFT_2450 ZP_05392450.1 255525514Clostridium carboxidivorans P7 CcarbDRAFT_1084 ZP_05391084.1 255524124Clostridium carboxidivorans P7 RnfC EDK33306.1 146346770 Clostridiumkluyveri RnfD EDK33307.1 146346771 Clostridium kluyveri RnfG EDK33308.1146346772 Clostridium kluyveri RnfE EDK33309.1 146346773 Clostridiumkluyveri RnfA EDK33310.1 146346774 Clostridium kluyveri RnfB EDK33311.1146346775 Clostridium kluyveri CLJU_c11410 (RnfB) ADK14209.1 300434442Clostridium ljungdahlii CLJU_c11400 (RnfA) ADK14208.1 300434441Clostridium ljungdahlii CLJU_c11390 (RnfE) ADK14207.1 300434440Clostridium ljungdahlii CLJU_c11380 (RnfG) ADK14206.1 300434439Clostridium ljungdahlii CLJU_c11370 (RnfD) ADK14205.1 300434438Clostridium ljungdahlii CLJU_c11360 (RnfC) ADK14204.1 300434437Clostridium ljungdahlii MOTH_1518 (NfnA) YP_430370.1 83590361 Moorellathermoacetica MOTH_1517(NfnB) YP_430369.1 83590360 Moorellathermoacetica CHY_1992 (NfnA) YP_360811.1 78045020 Carboxydothermushydrogenoformans CHY_1993 (NfnB) YP_360812.1 78044266 Carboxydothermushydrogenoformans CLJU_c37220 (NfnAB) YP_003781850.1 300856866Clostridium ljungdahliiFIG. 1, Step I—Formate Dehydrogenase

Formate dehydrogenase (FDH) catalyzes the reversible transfer ofelectrons from formate to an acceptor. Enzymes with FDH activity utilizevarious electron carriers such as, for example, NADH (EC 1.2.1.2), NADPH(EC 1.2.1.43), quinols (EC 1.1.5.6), cytochromes (EC 1.2.2.3) andhydrogenases (EC 1.1.99.33). FDH enzymes have been characterized fromMoorella thermoacetica (Andreesen and Ljungdahl, J Bacteriol 116:867-873(1973); Li et al., J Bacteriol 92:405-412 (1966); Yamamoto et al., JBiol Chem. 258:1826-1832 (1983). The loci, Moth_2312 is responsible forencoding the alpha subunit of formate dehydrogenase while the betasubunit is encoded by Moth_2314 (Pierce et al., Environ Microbiol(2008)). Another set of genes encoding formate dehydrogenase activitywith a propensity for CO₂ reduction is encoded by Sfum_2703 through Sfum2706 in Syntrophobacter fumaroxidans (de Bok et al., Eur J Biochem.270:2476-2485 (2003)); Reda et al., PNAS 105:10654-10658 (2008)). Asimilar set of genes presumed to carry out the same function are encodedby CHY_0731, CHY_0732, and CHY_0733 in C. hydrogenoformans (Wu et al.,PLoS Genet. 1:e65 (2005)). Formate dehydrogenases are also found manyadditional organisms including C. carboxidivorans P7, Bacillusmethanolicus, Burkholderia stabilis, Moorella thermoacetica ATCC 39073,Candida boidinii, Candida methylica, and Saccharomyces cerevisiae S288c.The soluble formate dehydrogenase from Ralstonia eutropha reduces NAD⁺(fdsG, -B, -A, -C, -D) (Oh and Bowien, 1998)

Protein GenBank ID GI Number Organism Moth_2312 YP_431142 148283121Moorella thermoacetica Moth_2314 YP_431144 83591135 Moorellathermoacetica Sfum_2703 YP_846816.1 116750129 Syntrophobacterfumaroxidans Sfum_2704 YP_846817.1 116750130 Syntrophobacterfumaroxidans Sfum_2705 YP_846818.1 116750131 Syntrophobacterfumaroxidans Sfum_2706 YP_846819.1 116750132 Syntrophobacterfumaroxidans CHY_0731 YP_359585.1 78044572 Carboxydothermushydrogenoformans CHY_0732 YP_359586.1 78044500 Carboxydothermushydrogenoformans CHY_0733 YP_359587.1 78044647 Carboxydothermushydrogenoformans CcarbDRAFT_0901 ZP_05390901.1 255523938 Clostridiumcarboxidivorans P7 CcarbDRAFT_4380 ZP_05394380.1 255527512 Clostridiumcarboxidivorans P7 fdhA, MGA3_06625 EIJ82879.1 387590560 Bacillusmethanolicus MGA3 fdhA, PB1_11719 ZP_10131761.1 387929084 Bacillusmethanolicus PB1 fdhD, MGA3_06630 EIJ82880.1 387590561 Bacillusmethanolicus MGA3 fdhD, PB1_11724 ZP_10131762.1 387929085 Bacillusmethanolicus PB1 fdh ACF35003. 194220249 Burkholderia stabilis FDH1AAC49766.1 2276465 Candida boidinii Fdh CAA57036.1 1181204 Candidamethylica FDH2 P0CF35.1 294956522 Saccharomyces cerevisiae S288c FDH1NP_015033.1 6324964 Saccharomyces cerevisiae S288cFIG. 1, Step J—Methanol Dehydrogenase

NAD+ dependent methanol dehydrogenase enzymes (EC 1.1.1.244) catalyzethe conversion of methanol and NAD+ to formaldehyde and NADH. An enzymewith this activity was first characterized in Bacillus methanolicus(Heggeset, et al., Applied and Environmental Microbiology,78(15):5170-5181 (2012)). This enzyme is zinc and magnesium dependent,and activity of the enzyme is enhanced by the activating enzyme encodedby act (Kloosterman et al, J Biol Chem 277:34785-92 (2002)). AdditionalNAD(P)+ dependent enzymes can be identified by sequence homology.Methanol dehydrogenase enzymes utilizing different electron acceptorsare also known in the art. Examples include cytochrome dependent enzymessuch as mxaIF of the methylotroph Methylobacterium extorquens (Nunn etal, Nucl Acid Res 16:7722 (1988)). Methanol dehydrogenase enzymes ofmethanotrophs such as Methylococcus capsulatis function in a complexwith methane monooxygenase (MMO) (Myronova et al, Biochem 45:11905-14(2006)). Methanol can also be oxidized to formaldehyde by alcoholoxidase enzymes such as methanol oxidase (EC 1.1.3.13) of Candidaboidinii (Sakai et al, Gene 114: 67-73 (1992)).

Protein GenBank ID GI Number Organism mdh, MGA3_17392 EIJ77596.1387585261 Bacillus methanolicus MGA3 mdh2, MGA3_07340 EIJ83020.1387590701 Bacillus methanolicus MGA3 mdh3, MGA3_10725 EIJ80770.1387588449 Bacillus methanolicus MGA3 act, MGA3_09170 EIJ83380.1387591061 Bacillus methanolicus MGA3 mdh, PB1_17533 ZP_10132907.1387930234 Bacillus methanolicus PB1 mdh1, PB1_14569 ZP_10132325.1387929648 Bacillus methanolicus PB1 mdh2, PB1_12584 ZP_10131932.1387929255 Bacillus methanolicus PB1 act, PB1_14394 ZP_10132290.1387929613 Bacillus methanolicus PB1 BFZC1_05383 ZP_07048751.1 299535429Lysinibacillus fusiformis BFZC1_20163 ZP_07051637.1 299538354Lysinibacillus fusiformis Bsph_4187 YP_001699778.1 169829620Lysinibacillus sphaericus Bsph_1706 YP_001697432.1 169827274Lysinibacillus sphaericus MCA0299 YP_112833.1 53802410 Methylococcuscapsulatis MCA0782 YP_113284.1 53804880 Methylococcus capsulatis mxaIYP_002965443.1 240140963 Methylobacterium extorquens mxaF YP_002965446.1240140966 Methylobacterium extorquens AOD1 AAA34321.1 170820 CandidaboidiniiFIG. 1, Step K—Spontaneous or Formaldehyde Activating Enzyme

The conversion of formaldehyde and THF to methylenetetrahydrofolate canoccur spontaneously. It is also possible that the rate of this reactioncan be enhanced by a formaldehyde activating enzyme. A formaldehydeactivating enzyme (Fae) has been identified in Methylobacteriumextorquens AM1 which catalyzes the condensation of formaldehyde andtetrahydromethanopterin to methylene tetrahydromethanopterin (Vorholt,et al., J. Bacteriol., 182(23), 6645-6650 (2000)). It is possible that asimilar enzyme exists or can be engineered to catalyze the condensationof formaldehyde and tetrahydrofolate to methylenetetrahydrofolate.Homologs exist in several organisms including Xanthobacter autotrophicusPy2 and Hyphomicrobium denitrificans ATCC 51888.

Protein GenBank ID GI Number Organism MexAM1_META1p1766 Q9FA38.317366061 Methylobacterium extorquens AM1 Xaut_0032 YP_001414948.1154243990 Xanthobacter autotrophicus Py2 Hden_1474 YP_003755607.1300022996 Hyphomicrobium denitrificans ATCC 51888FIG. 1, Step L—Formaldehyde Dehydrogenase

Oxidation of formaldehyde to formate is catalyzed by formaldehydedehydrogenase. An NAD+ dependent formaldehyde dehydrogenase enzyme isencoded by fdhA of Pseudomonas putida (Ito et al, J Bacteriol 176:2483-2491 (1994)). Additional formaldehyde dehydrogenase enzymes includethe NAD+ and glutathione independent formaldehyde dehydrogenase fromHyphomicrobium zavarzinii (Jerome et al, Appl Microbiol Biotechnol77:779-88 (2007)), the glutathione dependent formaldehyde dehydrogenaseof Pichia pastoris (Sunga et al, Gene 330:39-47 (2004)) and the NAD(P)+dependent formaldehyde dehydrogenase of Methylobacter marinus (Speer etal, FEMS Microbiol Lett, 121(3):349-55 (1994)).

Protein GenBank ID GI Number Organism fdhA P46154.3 1169603 Pseudomonasputida faoA CAC85637.1 19912992 Hyphomicrobium zavarzinii Fld1CCA39112.1 328352714 Pichia pastoris Fdh P47734.2 221222447Methylobacter marinus

In addition to the formaldehyde dehydrogenase enzymes listed above,alternate enzymes and pathways for converting formaldehyde to formateare known in the art. For example, many organisms employglutathione-dependent formaldehyde oxidation pathways, in whichformaldehyde is converted to formate in three steps via theintermediates S-hydroxymethylglutathione and S-formylglutathione(Vorholt et al, J Bacteriol 182:6645-50 (2000)). The enzymes of thispathway are S-(hydroxymethyl)glutathione synthase (EC 4.4.1.22),glutathione-dependent formaldehyde dehydrogenase (EC 1.1.1.284) andS-formylglutathione hydrolase (EC 3.1.2.12).

FIG. 1, Step M—Spontaneous or S-(Hydroxymethyl)Glutathione Synthase

While conversion of formaldehyde to S-hydroxymethylglutathione can occurspontaneously in the presence of glutathione, it has been shown byGoenrich et al (Goenrich, et al., J Biol Chem 277(5); 3069-72 (2002))that au enzyme from Paracoccus denitrificans can accelerate thisspontaneous condensation reaction. The enzyme catalyzing the conversionof formaldehyde and glutathione was purified and namedglutathione-dependent formaldehyde-activating enzyme (Gfa). The geneencoding it, which was named gfa, is located directly upstream of thegene for glutathione-dependent formaldehyde dehydrogenase, whichcatalyzes the subsequent oxidation of S-hydroxymethylglutathione.Putative proteins with sequence identity to Gfa from P. denitrificansare present also in Rhodobacter sphaeroides, Sinorhizobium meliloti, andMesorhizobium loti.

Protein GenBank ID GI Number Organism Gfa Q51669.3 38257308 Paracoccusdenitrificans Gfa ABP71667.1 145557054 Rhodobacter sphaeroides ATCC17025 Gfa Q92WX6.1 38257348 Sinorhizobium meliloti 1021 Gfa Q98LU4.238257349 Mesorhizobium loti MAFF303099FIG. 1, Step N—Glutathione-Dependent Formaldehyde Dehydrogenase

Glutathione-dependent formaldehyde dehydrogenase (GS-FDH) belongs to thefamily of class III alcohol dehydrogenases. Glutathione and formaldehydecombine non-enzymatically to form hydroxymethylglutathione, the truesubstrate of the GS-FDH catalyzed reaction. The product,S-formylglutathione, is further metabolized to formic acid.

Protein GenBank ID GI Number Organism frmA YP_488650.1 388476464Escherichia coli K-12 MG1655 SFA1 NP_010113.1 6320033 Saccharomycescerevisiae S288c flhA AAC44551.1 1002865 Paracoccus denitrificans adhIAAB09774.1 986949 Rhodobacter sphaeroidesFIG. 1, Step O—S-Formylglutathione Hydrolase

S-formylglutathione hydrolase is a glutathione thiol esterase found inbacteria, plants and animals. It catalyzes conversion ofS-formylglutathione to formate and glutathione. The fghA gene of P.denitrificans is located in the same operon with gfa and flhA, two genesinvolved in the oxidation of formaldehyde to formate in this organism.In E. coli, FrmB is encoded in an operon with FrmR and FrmA, which areproteins involved in the oxidation of formaldehyde. YeiG of E. coli is apromiscuous serine hydrolase; its highest specific activity is with thesubstrate S-formylglutathione.

Protein GenBank ID GI Number Organism frmB NP_414889.1 16128340Escherichia coli K-12 MG1655 yeiG AAC75215.1 1788477 Escherichia coliK-12 MG1655 fghA AAC44554.1 1002868 Paracoccus denitrificans

4.2 Example II—Enhanced Yield of 1,2-Propanediol and/or n-Propanol,1,3-Propanediol and/or Glycerol from Carbohydrates Using Methanol

Exemplary methanol metabolic pathways for enhancing the availability ofreducing equivalents are provided in FIG. 1.

1,2-propanediol and/or n-propanol can be achieved in a recombinantorganism by the pathway shown in FIG. 2. Exemplary enzymes for theconversion of glucose to 1,2-propanediol and/or n-propanol by this routeinclude 2A) a methylglyoxal synthase; 2B) a methylglyoxal reductase(acetol-forming); 2C) an acetol reductase; 2D) a methylglyoxal reductase(lactaldehyde-forming); 2E) a lactaldehyde reductase; 2F) a1,2-propanediol dehydratase; and 2G) a propanal reductase.1,2-propanediol production can be carried out by 2A, 2B and 2C; or 2A,2D and 2E. n-propanol production can be carried out by 2A, 2B, 2C, 2Fand 2G; or 2A, 2D, 2E, 2F and 2G.

FIG. 2 depicts two pathways for converting dihydroxyacetone phosphate to1,2-propanediol or n-propanol. Both pathways require the initialformation of methylglyoxal from dihydroxyacetone phosphate bymethylglyoxal synthase. Methylglyoxal can be subsequently reduced toform acetol or lactaldehyde by methylglyoxal reductase (acetol-forming)or methylglyoxal reductase (lactaldehyde-forming). Further reduction ofacetol or lactaldehyde yields 1,2-propanediol. An enzyme with1,2-propanediol dehydratase activity is required to dehydrate1,2-propanediol to propanal. Propanal is further reduced to propanol byan aldehyde reductase or alcohol dehydrogenase.

Enzyme candidates for converting dihydroxyacetone phosphate to1,2-propanediol and propanol are described in further detail below.

FIG. 2, Step A—Methylglyoxal Synthase

Methylglyoxal is formed from dihydroxyacetone phosphate by methylglyoxalsynthase (MGS, EC 4.2.3.3). This activity is encoded by mgs in E. coli(Altaras and Cameron., Appl Env Microbiol., 65:1180-1185 (1999)). Theintroduction of exogenous MGS in yeast has been shown to result in theproduction of low levels of 1,2-propanediol (Hoffman. M. L., 1999,Metabolic engineering of 1,2-propanediol production in Saccharomycescerevisiae. Ph.D. Dissertation, University of Wisconsin-Madison).Subsequent introduction of a glycerol dehydrogenase doubled the amountof propanediol formed. MGS enzymes from Thermus sp. GH5 and Clostridiumacetobutylicum were cloned, expressed and characterized in E. coli(Pazhang et al, Appl Biochem Biotechnol 162:1519-28 (2010); Huang et al,Appl Env Microbiol 65:3244-7 (1999)).

Protein GenBank ID GI Number Organism Mgs AAC74049.2 87081809Escherichia coli EU744585.1:1 . . . 399 ACE81430.1 190663729 Thermus sp.GH5 mgsA NP_348231.1 15894882 Clostridium acetobutylicumFIG. 2, Step B—Methylglyoxal Reductase (Acetol-Forming)

Methylglyoxal reductase can alternately be reduced to form acetol by anenzyme that converts aldehydes to alcohols. The aldehyde dehydrogenaseenzyme of Leishmania donovani exhibits methylglyoxal reductase (acetolforming) activity (Rath et al, Gene 429:1-9 (2009)). Exemplary genesinclude alrA encoding a medium-chain alcohol dehydrogenase for C2-C14(Tani et al., Appl. Environ. Microbiol. 66:5231-5235 (2000)), ADH2 fromSaccharomyces cerevisiae (Atsumi et al., Nature 451:86-89 (2008)), yqhDfrom E. coli which has preference for molecules longer than C(3)(Sulzenbacher et al., 342:489-502 (2004)), and bdh I and bdh II from C.acetobutylicum which converts butyryaldehyde into butanol (Walter etal., 174:7149-7158 (1992)). YqhD catalyzes the reduction of a wide rangeof aldehydes using NADPH as the cofactor (Perez et al., J Biol. Chem.283:7346-7353 (2008)). Another aldehyde reductase of E. coli with abroad substrate range is encoded by fucO. The adhA gene product fromZymomonas mobilisE has been demonstrated to have activity on a number ofaldehydes including formaldehyde, acetaldehyde, propionaldehyde,butyraldehyde, and acrolein (Kinoshita et al., Appl Microbiol Biotechnol22:249-254 (1985)). Additional aldehyde reductase candidates are encodedby bdh in C. saccharoperbutylacetonicum and Cbei_1722, Cbei_2181 andCbei_2421 in C. beijerinckii.

Protein GenBank ID GI Number Organism alr ABP24363.1 145025448Leishmania donovani alrA BAB12273.1 9967138 Acinetobacter sp. strain M-1ADH2 NP_014032.1 6323961 Saccharomyces cerevisiae yqhD NP_417484.116130909 Escherichia coli fucO NP_417279.1 16130706 Escherichia coli bdhI NP_349892.1 15896543 Clostridium acetobutylicum bdh II NP_349891.115896542 Clostridium acetobutylicum adhA YP_162971.1 56552132 Zymomonasmobilis bdh BAF45463.1 124221917 Clostridium saccharoperbutylacetonicumCbei_1722 YP_001308850 150016596 Clostridium beijerinckii Cbei_2181YP_001309304 150017050 Clostridium beijerinckii Cbei_2421 YP_001309535150017281 Clostridium beijerinckii

Enzymes exhibiting 4-hydroxybutyrate dehydrogenase activity (EC1.1.1.61) also fall into this category. Such enzymes have beencharacterized in Ralstonia eutropha (Bravo et al., J Forens Sci,49:379-387 (2004)), Clostridium kluyveri (Wolff et al., Protein Expr.Purif: 6:206-212 (1995)) and Arabidopsis thaliana (Breitkreuz et al., JBiol Chem, 278:41552-41556 (2003)). The A. thaliana enzyme was clonedand characterized in yeast (Breitkreuz et al., J. Biol. Chem.278:41552-41556 (2003)). Yet another gene is the alcohol dehydrogenaseadhI from Geobacillus thermoglucosidasius (Jeon et al., J Biotechnol135:127-133 (2008)).

Protein GenBank ID GI Number Organism 4hbd YP_726053.1 113867564Ralstonia eutropha H16 4hbd L21902.1 146348486 Clostridium kluyveri DSM555 4hbd Q94B07 75249805 Arabidopsis thaliana adhI AAR91477.1 40795502Geobacillus thermoglucosidasius

Another exemplary enzyme is methylmalonate semialdehyde reductase, alsoknown as 3-hydroxyisobutyrate dehydrogenase (EC 1.1.1.31). This enzymeparticipates in valine, leucine and isoleucine degradation and has beenidentified in bacteria, eukaryotes, and mammals. The enzyme encoded byP84067 from Thermus thermophilus HB8 has been structurally characterized(Lokanath et al., J Mol Biol, 352:905-17 (2005)). The reversibility ofthe human 3-hydroxyisobutyrate dehydrogenase was demonstrated usingisotopically-labeled substrate (Manning et al., Biochem J, 231:481-4(1985)). Additional genes encoding this enzyme include 3hidh in Homosapiens (Hawes et al., Methods Enzymol, 324:218-228 (2000)) andOryctolagus cuniculus (Hawes et al., supra; Chowdhury et al., Biosci.Biotechnol Biochem. 60:2043-2047 (1996)), mmsB in Pseudomonas aeruginosaand Pseudomonas putida, and dhat in Pseudomonas putida (Aberhart et al.,J Chem. Soc. [Perkin 1] 6:1404-1406 (1979); Chowdhury et al., Biosci.Biotechnol Biochem. 60:2043-2047 (1996); Chowdhury et al., Biosci.Biotechnol Biochem. 67:438-441 (2003)). Several 3-hydroxyisobutyratedehydrogenase enzymes have been characterized in the reductivedirection, including mmsB from Pseudomonas aeruginosa (Gokarn et al.,U.S. Pat. No. 739,676, (2008)) and mmsB from Pseudomonas putida.

Protein GenBank ID GI Number Organism P84067 P84067 75345323 Thermusthermophilus 3hidh P31937.2 12643395 Homo sapiens 3hidh P32185.1 416872Oryctolagus cuniculus mmsB NP_746775.1 26991350 Pseudomonas putida mmsBP28811.1 127211 Pseudomonas aeruginosa dhat Q59477.1 2842618 PseudomonasputidaFIG. 2, Step C—Acetol Reductase

The ketone of acetol is further reduced to an alcohol, forming1,2-propanediol, by an enzyme with acetol reductase activity. Thealdehyde reductase alr of Leishmania donovani has been shown to catalyzethis reaction (Rath et al, Gene 429:1-9 (2009)). Glycerol dehydrogenaseenzymes, including gldA of E. coli and dhaD of Klebsiella pneumonia,also convert acetol to 1,2-PDO (Lee and Whitesides, J Org Chem, 51:25-36(1986); Altaras and Cameron, Biotechnol Prog 16:940-46 (2000)) Theenzyme candidates described previously for catalyzing the reduction ofmethylglyoxal to acetol or lactaldehyde are also suitable acetolreductase enzyme candidates.

Protein GenBank ID GI Number Organism alr ABP24363.1 145025448Leishmania donovani gldA AAC76927.2 87082352 Escherichia coli dhaDABO15720.1 126513217 Klebsiella pneumoniaeFIG. 2, Step D—Methylglyoxal Reductase (Lactaldehyde-Forming);

The conversion of methylglyoxal to lactaldehyde is catalyzed by anenzyme with methylglyoxal reductase (lactaldehyde-forming) activity. Anumber of alcohol dehydrogenase enzymes are suitable for catalyzing thisreaction, including alcohol dehydrogenase (EC 1.1.1.1; 1.1.1.2),aldehyde reductase (EC 1.1.1.21), methylglyoxal reductase (EC 1.1.1.78;1.1.1.283), glycerol dehydrogenase (EC 1.1.1.6), and others. Exemplarygenes with methylglyoxal reductase (lactaldehyde-forming) activityinclude the glycerol dehydrogenase genes gldA of E. coli and dhaD ofKlebsiella pneumoniae (Altaras and Cameron., Appl Env Microbiol.,65:1180-1185 (1999)). These genes were successfully employed in 1,2-PDOproduction pathways. Another enzyme with this activity is encoded byGRE2 of Saccharomyces cerevisiae (Chen et al, Yeast, 20:545-54 (2003)).Additional candidates include malate dehydrogenase (mdh) and lactatedehydrogenase (ldhA) of E. coli. The lactate dehydrogenase fromRalstonia eutropha has been shown to demonstrate high activities on2-ketoacids of various chain lengths including lactate, 2-oxobutyrate,2-oxopentanoate and 2-oxoglutarate (Steinbuchel et al., Eur. J. Biochem.130:329-334 (1983)). Conversion of alpha-ketoadipate intoalpha-hydroxyadipate can be catalyzed by 2-ketoadipate reductase, anenzyme reported to be found in rat and in human placenta (Suda et al.,Arch. Biochem. Biophys. 176:610-620 (1976); Suda et al., Biochem.Biophys. Res. Commun. 77:586-591 (1977)). An additional oxidoreductaseis the mitochondrial 3-hydroxybutyrate dehydrogenase (bdh) from thehuman heart which has been cloned and characterized (Marks et al., J.Biol. Chem. 267:15459-15463 (1992)). Alcohol dehydrogenase enzymes of C.beijerinckii (Ismaiel et al., J. Bacteriol. 175:5097-5105 (1993)) and T.brockii (Lamed et al., Biochem. J. 195:183-190 (1981); Peretz et al.,Biochemistry. 28:6549-6555 (1989)) convert acetone to isopropanol.Methyl ethyl ketone reductase catalyzes the reduction of MEK to2-butanol. Exemplary MEK reductase enzymes can be found in Rhodococcusruber (Kosjek et al., Biotechnol Bioeng. 86:55-62 (2004)) and Pyrococcusfuriosus (van der et al., Eur. J. Biochem. 268:3062-3068 (2001)).

Protein GenBank ID GI Number Organism gldA AAC76927.2 87082352Escherichia coli dhaD ABO15720.1 126513217 Klebsiella pneumoniae GRE2Q12068.1 57013849 Saccharomyces cerevisiae Mdh AAC76268.1 1789632Escherichia coli ldhA NP_415898.1 16129341 Escherichia coli LdhYP_725182.1 113866693 Ralstonia eutropha Bdh AAA58352.1 177198 Homosapiens Adh AAA23199.2 60592974 Clostridium beijerinckii NRRL B593 AdhP14941.1 113443 Thermoanaerobacter brockii HTD4 Sadh CAD36475 21615553Rhodococcus ruber adhA AAC25556 3288810 Pyrococcus furiosusFIG. 2, Step E—Lactaldehyde Reductase

Aldehyde reductase enzymes are required to convert lactaldehyde to1,2-PDO. The aldehyde reductase encoded by fucO of E. coli convertsS-lactaldehyde to 1,2-PDO (Altaras and Cameron., Appl Env Microbiol.,65:1180-1185 (1999)). Gene candidates in Saccharomyces cerevisiaeinclude the aldehyde reductases GRE3, ALD2-6 and HFD1, glyoxylatereductases GOR1 and YPL113C and glycerol dehydrogenase GCY1 (WO2011/022651A1). The enzyme candidates described previously forcatalyzing the reduction of methylglyoxal to acetol or lactaldehyde arealso suitable lactaldehyde reductase enzyme candidates.

Protein GenBank ID GI Number Organism fucO NP_417279.1 16130706Escherichia coli GRE3 P38715.1 731691 Saccharomyces cerevisiae ALD2CAA89806.1 825575 Saccharomyces cerevisiae ALD3 NP_013892.1 6323821Saccharomyces cerevisiae ALD4 NP_015019.1 6324950 Saccharomycescerevisiae ALD5 NP_010996.2 330443526 Saccharomyces cerevisiae ALD6ABX39192.1 160415767 Saccharomyces cerevisiae HFD1 Q04458.1 2494079Saccharomyces cerevisiae GOR1 NP_014125.1 6324055 Saccharomycescerevisiae YPL113C AAB68248.1 1163100 Saccharomyces cerevisiae GCY1CAA99318.1 1420317 Saccharomyces cerevisiaeFIG. 2, Step F—1,2-Propanediol Dehydratase;

Dehydration of 1,2-propanediol to propanal is catalyzed by a dioldehydratase enzyme with 1,2-propanediol dehydratase activity. Exemplarydiol dehydratase enzymes include propanediol dehydratase (EC 4.2.1.28),glycerol dehydratase (EC 4.2.1.30) and dihydroxy-acid dehydratase (EC4.2.1.9). Enzymes may require adenosylcobalamin (B12) as a cofactor orbe B12-independent. B12-dependent diol dehydratases contain alpha, betaand gamma subunits, which are all required for enzyme function.

Diol dehydratase or propanediol dehydratase enzymes (EC 4.2.1.28)capable of converting the secondary diol 2,3-butanediol to 2-butanonewould be excellent candidates for this transformation. Exemplary1,2-propanediol dehydratase enzyme candidates are found in Klebsiellapneumoniae (Toraya et al., Biochem. Biophys. Res. Commun. 69:475-480(1976); Tobimatsu et al., Biosci. Biotechnol Biochem. 62:1774-1777(1998)), Salmonella typhimurium (Bobik et al., J. Bacteriol.179:6633-6639 (1997)), Klebsiella oxytoca (Tobimatsu et al., J Biol.Chem. 270:7142-7148 (1995)) and Lactobacillus collinoides (Sauvageot etal., FEMS Microbiol Lett. 209:69-74 (2002)). Methods for isolating dioldehydratase gene candidates in other organisms are well known in the art(e.g. U.S. Pat. No. 5,686,276).

Protein GenBank ID GI Number Organism pddC AAC98386.1 4063704 Klebsiellapneumoniae pddB AAC98385.1 4063703 Klebsiella pneumoniae pddA AAC98384.14063702 Klebsiella pneumoniae pduC AAB84102.1 2587029 Salmonellatyphimurium pduD AAB84103.1 2587030 Salmonella typhimurium pduEAAB84104.1 2587031 Salmonella typhimurium pddA BAA08099.1 868006Klebsiella oxytoca pddB BAA08100.1 868007 Klebsiella oxytoca pddCBAA08101.1 868008 Klebsiella oxytoca pduC CAC82541.1 18857678Lactobacillus collinoides pduD CAC82542.1 18857679 Lactobacilluscollinoides pduE CAD01091.1 18857680 Lactobacillus collinoides

Enzymes in the glycerol dehydratase family (EC 4.2.1.30) can also beused to dehydrate 1,2-propanediol. Exemplary gene candidates encoded bygldABC and dhaB123 in Klebsiella pneumoniae (World Patent WO2008/137403) and (Toraya et al., Biochem. Biophys. Res. Commun.69:475-480 (1976)), dhaBCE in Clostridium pasteuranum (Macis et al.,FEMS Microbiol Lett. 164:21-28 (1998)) and dhaBCE in Citrobacterfreundii (Seyfried et al., J Bacteriol. 178:5793-5796 (1996)). Variantsof the B12-dependent diol dehydratase from K. pneumoniae with 80- to336-fold enhanced activity were recently engineered by introducingmutations in two residues of the beta subunit (Qi et al., J. Biotechnol.144:43-50 (2009)). Diol dehydratase enzymes with reduced inactivationkinetics were developed by DuPont using error-prone PCR (WO2004/056963).

Protein GenBank ID GI Number Organism gldA AAB96343.1 1778022 Klebsiellapneumonia gldB AAB96344.1 1778023 Klebsiella pneumonia gldC AAB96345.11778024 Klebsiella pneumoniae dhaB1 ABR78884.1 150956854 Klebsiellapneumoniae dhaB2 ABR78883.1 150956853 Klebsiella pneumoniae dhaB3ABR78882.1 150956852 Klebsiella pneumoniae dhaB AAC27922.1 3360389Clostridium pasteuranum dhaC AAC27923.1 3360390 Clostridium pasteuranumdhaE AAC27924.1 3360391 Clostridium pasteuranum dhaB P45514.1 1169287Citrobacter freundii dhaC AAB48851.1 1229154 Citrobacter freundii dhaEAAB48852.1 1229155 Citrobacter freundii

If a B12-dependent diol dehydratase is utilized, heterologous expressionof the corresponding reactivating factor is recommended. B12-dependentdiol dehydratases are subject to mechanism-based suicide activation bysubstrates and some downstream products. Inactivation, caused by a tightassociation with inactive cobalamin, can be partially overcome by dioldehydratase reactivating factors in an ATP-dependent process.Regeneration of the B12 cofactor requires an additional ATP. Dioldehydratase regenerating factors are two-subunit proteins. Exemplarycandidates are found in Klebsiella oxytoca (Mori et al., J Biol. Chem.272:32034-32041 (1997)), Salmonella typhimurium (Bobik et al., JBacteriol. 179:6633-6639 (1997); Chen et al., J Bacteriol. 176:5474-5482(1994)), Lactobacillus collinoides (Sauvageot et al., FEMS MicrobiolLett. 209:69-74 (2002)), Klebsiella pneumonia (World Patent WO2008/137403).

Protein GenBank ID GI Number Organism ddrA AAC15871 3115376 Klebsiellaoxytoca ddrB AAC15872 3115377 Klebsiella oxytoca pduG AAB84105 16420573Salmonella typhimurium pduH AAD39008 16420574 Salmonella typhimuriumpduG YP_002236779 206579698 Klebsiella pneumonia pduH YP_002236778206579863 Klebsiella pneumonia pduG CAD01092 29335724 Lactobacilluscollinoides pduH AJ297723 29335725 Lactobacillus collinoides

B12-independent diol dehydratase enzymes utilize S-adenosylmethionine(SAM) as a cofactor and function under strictly anaerobic conditions.The glycerol dehydrogenase and corresponding activating factor ofClostridium butyricum, encoded by dhaB1 and dhaB2, have beenwell-characterized (O'Brien et al., Biochemistry 43:4635-4645 (2004);Raynaud et al., Proc. Natl. Acad. Sci U.S.A 100:5010-5015 (2003)). Thisenzyme was recently employed in a 1,3-propanediol overproducing strainof E. coli and was able to achieve very high titers of product (Tang etal., Appl. Environ. Microbiol. 75:1628-1634 (2009)). An additionalB12-independent diol dehydratase enzyme and activating factor fromRoseburia inulinivorans was shown to catalyze the conversion of2,3-butanediol to 2-butanone (US 2009/09155870).

Protein GenBank ID GI Number Organism dhaB1 AAM54728.1 27461255Clostridium butyricum dhaB2 AAM54729.1 27461256 Clostridium butyricumrdhtA ABC25539.1 83596382 Roseburia inulinivorans rdhtB ABC25540.183596383 Roseburia inulinivorans

Dihydroxy-acid dehydratase (DHAD, EC 4.2.1.9) is a B12-independentenzyme participating in branched-chain amino acid biosynthesis. In itsnative role, it converts 2,3-dihydroxy-3-methylvalerate to2-keto-3-methyl-valerate, a precursor of isoleucine. In valinebiosynthesis the enzyme catalyzes the dehydration of2,3-dihydroxy-isovalerate to 2-oxoisovalerate. The DHAD from Sulfolobussolfataricus has a broad substrate range and activity of a recombinantenzyme expressed in E. coli was demonstrated on a variety of aldonicacids (KIM et al., J. Biochem. 139:591-596 (2006)). The S. solfataricusenzyme is tolerant of oxygen unlike many diol dehydratase enzymes.Substrate (1) has not been tested to date. The E. coli enzyme, encodedby ilvD, is sensitive to oxygen, which inactivates its iron-sulfurcluster (Flint et al., J. Biol. Chem. 268:14732-14742 (1993)). Similarenzymes have been characterized in Neurospora crassa (Altmiller et al.,Arch. Biochem. Biophys. 138:160-170 (1970)) and Salmonella typhimurium(Armstrong et al., Biochim. Biophys. Acta 498:282-293 (1977)).

Protein GenBank ID GI Number Organism ilvD NP_344419.1 15899814Sulfolobus solfataricus ilvD AAT48208.1 48994964 Escherichia coli ilvDNP_462795.1 16767180 Salmonella typhimurium ilvD XP_958280.1 85090149Neurospora crassaFIG. 2, Step G—Propanal Reductase

The alcohol dehydrogenase enzyme candidates described above are alsosuitable for catalyzing the reduction of propanal to propanol.

4.3 Example III—Enhanced Yield of 1,3-Propanediol and/or Glycerol fromCarbohydrates Using Methanol

Exemplary methanol metabolic pathways for enhancing the availability ofreducing equivalents are provided in FIG. 1.

1,3-propanediol and/or glycerol production can be achieved in arecombinant organism by the pathway shown in FIG. 3. Exemplary enzymesfor the conversion of glucose to 1,3-propanediol and/or glycerol by thisroute include 3A) a glyceraldehyde-3-phosphate reductase; 3B) aglycerol-3-phosphate phosphatase or a glycerol kinase; 3C) a glyceroldehydratase; 3D) a 3-hydroxypropanal reductase; 3E) a dihydroxyacetonephosphate phosphatase or a dihydroxyacetone kinase; 3F) adihydroxyacetone reductase; and 3G) a dihydroxyacetone phosphatereductase. 1,3-propanediol production can be carried out by 3A, 3B, 3Cand 3D; 3G, 3B, 3C and 3D; or 3E, 3F, 3C and 3D. Glycerol production canbe carried out by 3A and 3B; 3G and 3B; or 3E and 3F.

The additional reducing equivalents obtained from the methanol metabolicpathways provided herein improve the yields of 1,3-propanediol andglycerol when utilizing a carbohydrate-based feedstock. 1,3-propanediolor glycerol production can be achieved in a recombinant organism byalternative pathways as shown in FIG. 3. In the first route,glyceraldehyde-3-phosphate is reduced to glycerol-3-phosphate.Glycerol-3-phosphate is subsequently dephosphorylated to glycerol byeither a phosphatase or a glycerol kinase enzyme. Glycerol can then besecreted as a product or further converted to 1,3-PDO by a dioldehydratase and a 3-hydroxypropanal reductase. Alternately, theglycerol-3-phosphate intermediate is formed by the reduction ofdihydroxyacetone phosphate. In yet another route, dihydroxyacetonephosphate is first dephosphorylated to dihydroxyacetone by a kinase orphosphatase. Dihydroxyacetone is then reduced to glycerol, which can besecreted or further converted to 1,3-PDO as described above.

Exemplary enzyme candidates for the transformations shown in FIG. 3 aredescribed below. The following table shows enzyme classes that canperform the steps depicted in FIG. 3.

Label Function Step 1.1.1.a Oxidoreductase (oxo to alcohol) A, D, F, G3.1.3.a Phosphatase B, E 2.7.2.a Kinase B, E 4.2.1.a Dehydratase CFIG. 3, Step A=Glyceraldehyde-3-Phosphate Reductase

Enzymes that reduce aldehydes to alcohols are suitable candidates forthe reduction of 3-hydroxypropanal and glyceraldehyde-3-phosphate.Exemplary genes encoding enzymes that catalyze the conversion of analdehyde to alcohol (i.e., alcohol dehydrogenase or equivalentlyaldehyde reductase) include alrA encoding a medium-chain alcoholdehydrogenase for C2-C14 (Tani et al., Appl. Environ. Microbiol.66:5231-5235 (2000)), yqhD and fucO from E. coli (Suizenbacher et al.,342:489-502 (2004)), and bdh I and bdh II from C. acetobutylicum whichconverts butyryaldehyde into butanol (Walter et al., 174:7149-7158(1992)). YqhD catalyzes the reduction of a wide range of aldehydes usingNADPH as the cofactor, with a preference for chain lengths longer thanC(3) (Sulzenbacher et al., 342:489-502 (2004); Perez et al., J Biol.Chem. 283:7346-7353 (2008)). The adhA gene product from ZymomonasmobilisE has been demonstrated to have activity on a number of aldehydesincluding formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde,and acrolein (Kinoshita et al., Appl Microbiol Biotechnol 22:249-254(1985)). Additional aldehyde reductase candidates are encoded by bdh inC. saccharoperbutylacetonicum and Cbei_1722, Cbei_2181 and Cbei_2421 inC. beijerinckii.

Protein GenBank ID GI Number Organism alrA BAB12273.1 9967138Acinetobacter sp. train M-1 ADH2 NP_014032.1 6323961 Saccharomycescerevisiae yqhD NP_417484.1 16130909 Escherichia coli fucO NP_417279.116130706 Escherichia coli bdh I NP_349892.1 15896543 Clostridiumacetobutylicum bdh II NP_349891.1 15896542 Clostridium acetobutylicumadhA YP_162971.1 56552132 Zymomonas mobilis Bdh BAF45463.1 124221917Clostridium saccharoperbutylacetonicum Cbei_1722 YP_001308850 150016596Clostridium beijerinckii Cbei_2181 YP_001309304 150017050 Clostridiumbeijerinckii Cbei_2421 YP_001309535 150017281 Clostridium beijerinckii

Aldehyde reductase gene candidates in Saccharomyces cerevisiae includethe aldehyde reductases GRE3, ALD2-6 and HFD1, glyoxylate reductasesGOR1 and YPL113C and glycerol dehydrogenase GCY1 (WO 2011/022651A1;Atsumi et al., Nature 451:86-89 (2008)). The enzyme candidates describedpreviously for catalyzing the reduction of methylglyoxal to acetol orlactaldehyde are also suitable lactaldehyde reductase enzyme candidates.

Protein GenBank ID GI Number Organism GRE3 P38715.1 731691 Saccharomycescerevisiae ALD2 CAA89806.1 825575 Saccharomyces cerevisiae ALD3NP_013892.1 6323821 Saccharomyces cerevisiae ALD4 NP_015019.1 6324950Saccharomyces cerevisiae ALD5 NP_010996.2 330443526 Saccharomycescerevisiae ALD6 ABX39192.1 160415767 Saccharomyces cerevisiae HFD1Q04458.1 2494079 Saccharomyces cerevisiae GOR1 NP_014125.1 6324055Saccharomyces cerevisiae YPL113C AAB68248.1 1163100 Saccharomycescerevisiae GCY1 CAA99318.1 1420317 Saccharomyces cerevisiae

Enzymes exhibiting 4-hydroxybutyrate dehydrogenase activity (EC1.1.1.61) also fall into this category. Such enzymes have beencharacterized in Ralstonia eutropha (Bravo et al., J Forens Sci,49:379-387 (2004)), Clostridium kluyveri (Wolff et al., Protein Expr.Purif. 6:206-212 (1995)) and Arabidopsis thaliana (Breitkreuz et al., JBiol Chem, 278:41552-41556 (2003)). The A. thaliana enzyme was clonedand characterized in yeast (Breitkreuz et al., J. Biol. Chem.278:41552-41556 (2003)). Yet another gene is the alcohol dehydrogenaseadhI from Geobacillus thermoglucosidasius (Jeon et al., J Biotechnol135:127-133 (2008)).

Protein GenBank ID GI Number Organism 4hbd YP_726053.1 113867564Ralstonia eutropha H16 4hbd L21902.1 146348486 Clostridium kluyveri DSM555 4hbd Q94B07 75249805 Arabidopsis thaliana adhI AAR91477.1 40795502Geobacillus thermoglucosidasius

Another exemplary enzyme is methylmalonate semialdehyde reductase, alsoknown as 3-hydroxyisobutyrate dehydrogenase (EC 1.1.1.31). This enzymeparticipates in valine, leucine and isoleucine degradation and has beenidentified in bacteria, eukaryotes, and mammals. The enzyme encoded byP84067 from Thermus thermophilus HB8 has been structurally characterized(Lokanath et al., J Mol Biol, 352:905-17 (2005)). The reversibility ofthe human 3-hydroxyisobutyrate dehydrogenase was demonstrated usingisotopically-labeled substrate (Manning et al., Biochem J, 231:481-4(1985)). Additional genes encoding this enzyme include 3hidh in Homosapiens (Hawes et al., Methods Enzymol, 324:218-228 (2000)) andOryctolagus cuniculus (Hawes et al., supra; Chowdhury et al., Biosci.Biotechnol Biochem. 60:2043-2047 (1996)), mmsB in Pseudomonas aeruginosaand Pseudomonas putida, and dhat in Pseudomonas putida (Aberhart et al.,J Chem. Soc. [Perkin 1] 6:1404-1406 (1979); Chowdhury et al., Biosci.Biotechnol Biochem. 60:2043-2047 (1996); Chowdhury et al., Biosci.Biotechnol Biochem. 67:438-441 (2003)). Several 3-hydroxyisobutyratedehydrogenase enzymes have been characterized in the reductivedirection, including mmsB from Pseudomonas aeruginosa (Gokarn et al.,U.S. Pat. No. 739,676, (2008)) and mmsB from Pseudomonas putida.

Protein GenBank ID GI Number Organism P84067 P84067 75345323 Thermusthermophilus 3hidh P31937.2 12643395 Homo sapiens 3hidh P32185.1 416872Oryctolagus cuniculus mmsB NP_746775.1 26991350 Pseudomonas putida mmsBP28811.1 127211 Pseudomonas aeruginosa dhat Q59477.1 2842618 Pseudomonasputida

Other suitable enzyme candidates for hydrolyzingglyceraldehydes-3-phosphate or dihydroxyacetone phosphate is3-phosphoglycerate phosphatase (EC 3.1.3.38), catalyzing the hydrolysisof 3PG to glycerate. The enzyme is found in plants and has a broadsubstrate range that includes phosphoenolpyruvate,ribulose-1,5-bisphosphate, dihydroxyacetone phosphate andglucose-6-phosphate (Randall et al., Plant Physiol 48:488-492 (1971);Randall et al., J Biol. Chem. 246:5510-5517 (1971)). Purified enzymefrom various plant sources has been characterized but a gene has notbeen associated with this enzyme to date. Another enzyme with3-phosphoglycerate phosphatase activity is the phosphoglyceratephosphatase (EC 3.1.3.20) from pig liver (Fallon et al., Biochim.Biophys. Acta 105:43-53 (1965)). The gene associated with this enzyme isnot available.

FIG. 3, Step B—Glycerol-3-Phosphate Phosphatase or Glycerol Kinase

Kinase or phosphotransferase enzymes in the EC class 2.7.2 transformcarboxylic acids to phosphonic acids with concurrent hydrolysis of oneATP. Such an enzyme is required to form dihydroxyacetone fromdihydroxyacetone phosphate and glycerol from glycerol-3-phosphate.Exemplary dihydroxyacetone kinases enzymes (EC 2.7.2.29) include DAKfrom Pichia angusta and dhaK from Citrobacter freundii (van der Klei etal, Curr Genet, 34:1-11 (1998); Daniel et al, J Bacteriol, 177:4392-401(1995)). The E. coli enzyme is encoded by dhaKLM (Bachler et al, EMBO J,24: 283-93 (2005)).

Protein GenBank ID GI Number Organism dhaK AAC74284.2 87081857Escherichia coli dhaL AAC74283.1 1787449 Escherichia coli dhaMNP_415717.1 16129162 Escherichia coli DAK AAC27705.1 3171001 Pichiaangusta dhaK AAB48843.1 493083 Citrobacter freundii

Glycerol kinase (EC 2.7.2.30) catalyzes the ATP-dependentphosphorylation of glycerol. Exemplary enzymes are encoded by glpK of E.coli (Pettegrew et al, J Biol Chem 263:135-139 (1988), Atlg80460 ofArabidopsis thaliana (Eastmond, Plant J 37:617-24 (2004)), glpK ofEnterococcus casseliflavus (Yeh et al, Biochem, 48:346-56 (2009)) andglpK of Haemophilus influenzae (Pawlyk and Pettigrew, Prot Expr Purif22:52-9 (2001)).

Protein GenBank ID GI Number Organism glpK AAC76908.1 1790361Escherichia coli At1g80460 BAH19502.1 222423040 Arabidopsis thalianaglpK O34153.3 3122148 Enterococcus casseliflavus glpK NP_438851.116272633 Haemophilus influenzae

The hydrolysis of glycerol-3-phosphate to glycerol is catalyzed byglycerol-3-phosphatase. Enzymes with this activity include theglycerol-1-phosphatase (EC 3.1.3.21) enzymes of Saccharomyces cerevisiae(GPP1 and GPP2), Candida albicans and Dunaleilla parva (Popp et al,Biotechnol Bioeng 100:497-505 (2008); Fan et al, FEMS Microbiol Lett245:107-16 (2005)). The D. parva gene has not been identified to date.

Protein GenBank ID GI Number Organism GPP1 DAA08494.1 285812595Saccharomyces cerevisiae GPP2 NP_010984.1 6320905 Saccharomycescerevisiae GPP1 XP_717809.1 68476319 Candida albicansFIG. 3, Step C—Glycerol Dehydratase

The dehydration of glycerol to 3-hydroxypropanal is catalyzed by a dioldehydratase enzyme with glycerol dehydratase activity. The enzymecandidates described above in Example II for propanediol dehydratase arealso applicable here. Exemplary diol dehydratase enzymes includepropanediol dehydratase (EC 4.2.1.28), glycerol dehydratase (EC4.2.1.30) and dihydroxy-acid dehydratase (EC 4.2.1.9). Enzymes mayrequire adenosylcobalamin (B12) as a cofactor or be B12-independent.B12-dependent diol dehydratases contain alpha, beta and gamma subunits,which are all required for enzyme function.

Diol dehydratase or propanediol dehydratase enzymes (EC 4.2.1.28)capable of converting the secondary diol 2,3-butanediol to 2-butanonewould be excellent candidates for this transformation. Exemplary1,2-propanediol dehydratase enzyme candidates are found in Klebsiellapneumoniae (Toraya et al., Biochem. Biophys. Res. Commun. 69:475-480(1976); Tobimatsu et al., Biosci. Biotechnol Biochem. 62:1774-1777(1998)), Salmonella typhimurium (Bobik et al., J Bacteriol.179:6633-6639 (1997)), Klebsiella oxytoca (Tobimatsu et al., J Biol.Chem. 270:7142-7148 (1995)) and Lactobacillus collinoides (Sauvageot etal., FEMS Microbiol Lett. 209:69-74 (2002)). Methods for isolating dioldehydratase gene candidates in other organisms are well known in the art(e.g. U.S. Pat. No. 5,686,276).

Protein GenBank ID GI Number Organism pddC AAC98386.1 4063704 Klebsiellapneumoniae pddB AAC98385.1 4063703 Klebsiella pneumoniae pddA AAC98384.14063702 Klebsiella pneumoniae pduC AAB84102.1 2587029 Salmonellatyphimurium pduD AAB84103.1 2587030 Salmonella typhimurium pduEAAB84104.1 2587031 Salmonella typhimurium pddA BAA08099.1 868006Klebsiella oxytoca pddB BAA08100.1 868007 Klebsiella oxytoca pddCBAA08101.1 868008 Klebsiella oxytoca pduC CAC82541.1 18857678Lactobacillus collinoides pduD CAC82542.1 18857679 Lactobacilluscollinoides pduE CAD01091.1 18857680 Lactobacillus collinoides

Enzymes in the glycerol dehydratase family (EC 4.2.1.30) can also beused to dehydrate 1,2-propanediol. Exemplary gene candidates encoded bygldABC and dhaB123 in Klebsiella pneumoniae (World Patent WO2008/137403) and (Toraya et al., Biochem. Biophys. Res. Commun.69:475-480 (1976)), dhaBCE in Clostridium pasteuranum (Macis et al.,FEMS Microbiol Lett. 164:21-28 (1998)) and dhaBCE in Citrobacterfreundii (Seyfried et al., J Bacteriol. 178:5793-5796 (1996)). Variantsof the B12-dependent diol dehydratase from K. pneumoniae with 80- to336-fold enhanced activity were recently engineered by introducingmutations in two residues of the beta subunit (Qi et al., J Biotechnol.144:43-50 (2009)). Diol dehydratase enzymes with reduced inactivationkinetics were developed by DuPont using error-prone PCR (WO2004/056963).

Protein GenBank ID GI Number Organism gldA AAB96343.1 1778022 Klebsiellapneumonia gldB AAB96344.1 1778023 Klebsiella pneumonia gldC AAB96345.11778024 Klebsiella pneumoniae dhaB1 ABR78884.1 150956854 Klebsiellapneumoniae dhaB2 ABR78883.1 150956853 Klebsiella pneumoniae dhaB3ABR78882.1 150956852 Klebsiella pneumoniae dhaB AAC27922.1 3360389Clostridium pasteuranum dhaC AAC27923.1 3360390 Clostridium pasteuranumdhaE AAC27924.1 3360391 Clostridium pasteuranum dhaB P45514.1 1169287Citrobacter freundii dhaC AAB48851.1 1229154 Citrobacter freundii dhaEAAB48852.1 1229155 Citrobacter freundii

If a B12-dependent diol dehydratase is utilized, heterologous expressionof the corresponding reactivating factor is recommended. B12-dependentdiol dehydratases are subject to mechanism-based suicide activation bysubstrates and some downstream products. Inactivation, caused by a tightassociation with inactive cobalamin, can be partially overcome by dioldehydratase reactivating factors in an ATP-dependent process.Regeneration of the B12 cofactor requires an additional ATP. Dioldehydratase regenerating factors are two-subunit proteins. Exemplarycandidates are found in Klebsiella oxytoca (Mori et al., J Biol. Chem.272:32034-32041 (1997)), Salmonella typhimurium (Bobik et al., JBacteriol. 179:6633-6639 (1997); Chen et al., J Bacteriol. 176:5474-5482(1994)), Lactobacillus collinoides (Sauvageot et al., FEMS MicrobiolLett. 209:69-74 (2002)), Klebsiella pneumonia (World Patent WO2008/137403).

Protein GenBank ID GI Number Organism ddrA AAC15871 3115376 Klebsiellaoxytoca ddrB AAC15872 3115377 Klebsiella oxytoca pduG AAB84105 16420573Salmonella typhimurium pduH AAD39008 16420574 Salmonella typhimuriumpduG YP_002236779 206579698 Klebsiella pneumonia pduH YP_002236778206579863 Klebsiella pneumonia pduG CAD01092 29335724 Lactobacilluscollinoides pduH AJ297723 29335725 Lactobacillus collinoides

B12-independent diol dehydratase enzymes utilize S-adenosylmethionine(SAM) as a cofactor and function under strictly anaerobic conditions.The glycerol dehydrogenase and corresponding activating factor ofClostridium butyricum, encoded by dhaB1 and dhaB2, have beenwell-characterized (O'Brien et al., Biochemistry 43:4635-4645 (2004);Raynaud et al., Proc. Natl. Acad. Sci U.S.A 100:5010-5015 (2003)). Thisenzyme was recently employed in a 1,3-propanediol overproducing strainof E. coli and was able to achieve very high titers of product (Tang etal., Appl. Environ. Microbiol. 75; 1628-1634 (2009)). An additionalB12-independent diol dehydratase enzyme and activating factor fromRoseburia inulinivorans was shown to catalyze the conversion of2,3-butanediol to 2-butanone (US 2009/09155870).

Protein GenBank ID GI Number Organism dhaB1 AAM54728.1 27461255Clostridium butyricum dhaB2 AAM54729.1 27461256 Clostridium butyricumrdhtA ABC25539.1 83596382 Roseburia inulinivorans rdhtB ABC25540.183596383 Roseburia inulinivorans

Dihydroxy-acid dehydratase (DHAD, EC 4.2.1.9) is a B12-independentenzyme participating in branched-chain amino acid biosynthesis. In itsnative role, it converts 2,3-dihydroxy-3-methylvalerate to2-keto-3-methyl-valerate, a precursor of isoleucine. In valinebiosynthesis the enzyme catalyzes the dehydration of2,3-dihydroxy-isovalerate to 2-oxoisovalerate. The DHAD from Sulfolobussolfataricus has a broad substrate range and activity of a recombinantenzyme expressed in E. coli was demonstrated on a variety of aldonicacids (KIM et al., J. Biochem. 139:591-596 (2006)), The S. solfataricusenzyme is tolerant of oxygen unlike many diol dehydratase enzymes.Substrate (1) has not been tested to date. The E. coli enzyme, encodedby ilvD, is sensitive to oxygen, which inactivates its iron-sulfurcluster (Flint et al., J. Biol. Chem. 268:14732-14742 (1993)). Similarenzymes have been characterized in Neurospora crassa (Altmiller et al.,Arch. Biochem. Biophys. 138:160-170 (1970)) and Salmonella typhimurium(Armstrong et al., Biochim. Biophys. Acta 498:282-293 (1977)).

Protein GenBank ID GI Number Organism ilvD NP_344419.1 15899814Sulfolobus solfataricus ilvD AAT48208.1 48994964 Escherichia coli ilvDNP_462795.1 16767180 Salmonella typhimurium ilvD XP_958280.1 85090149Neurospora crassa

FIG. 3, Step D—3-Hydroxypropanal Reductase Enzymes that reduce aldehydesto alcohols are suitable candidates for the reduction of3-hydroxypropanal and glyceraldehyde-3-phosphate. Exemplary genesencoding enzymes that catalyze the conversion of an aldehyde to alcohol(i.e., alcohol dehydrogenase or equivalently aldehyde reductase) includealrA encoding a medium-chain alcohol dehydrogenase for C2-C14 (Tani etal., Appl. Environ. Microbiol. 66:5231-5235 (2000)), yqhD and fucO fromE. coli (Sulzenbacher et al., 342:489-502 (2004)), and bdh I and bdh IIfrom C. acetobutylicum which converts butyryaldehyde into butanol(Walter et al., 174:7149-7158 (1992)). YqhD catalyzes the reduction of awide range of aldehydes using NADPH as the cofactor, with a preferencefor chain lengths longer than C(3) (Sulzenbacher et al., 342:489-502(2004); Perez et al., J Biol. Chem. 283:7346-7353 (2008)). The adhA geneproduct from Zymomonas mobilisE has been demonstrated to have activityon a number of aldehydes including formaldehyde, acetaldehyde,propionaldehyde, butyraldehyde, and acrolein (Kinoshita et al., ApplMicrobiol Biotechnol 22:249-254 (1985)). Additional aldehyde reductasecandidates are encoded by bdh in C. saccharoperbutylacetonicum andCbei_1722, Cbei_2181 and Cbei_2421 in C. beijerinckii.

Protein GenBank ID GI Number Organism alrA BAB12273.1 9967138Acinetobacter sp. strain M-1 ADH2 NP_014032.1 6323961 Saccharomycescerevisiae yqhD NP_417484.1 16130909 Escherichia coli fucO NP_417279.116130706 Escherichia coli bdh I NP_349892.1 15896543 Clostridiumacetobutylicum bdh II NP_349891.1 15896542 Clostridium acetobutylicumadhA YP_162971.1 56552132 Zymomonas mobilis bdh BAF45463.1 124221917Clostridium saccharoperbutylacetonicum Cbei_1722 YP_001308850 150016596Clostridium beijerinckii Cbei_2181 YP_001309304 150017050 Clostridiumbeijerinckii Cbei_2421 YP_001309535 150017281 Clostridium beijerinckii

Aldehyde reductase gene candidates in Saccharomyces cerevisiae includethe aldehyde reductases GRE3, ALD2-6 and HFD1, glyoxylate reductasesGOR1 and YPL113C and glycerol dehydrogenase GCY1 (WO 2011/022651A1;Atsumi et al., Nature 451:86-89 (2008)). The enzyme candidates describedpreviously for catalyzing the reduction of methylglyoxal to acetol orlactaldehyde are also suitable lactaldehyde reductase enzyme candidates.

Protein GenBank ID GI Number Organism GRE3 P38715.1 731691 Saccharomycescerevisiae ALD2 CAA89806.1 825575 Saccharomyces cerevisiae ALD3NP_013892.1 6323821 Saccharomyces cerevisiae ALD4 NP_015019.1 6324950Saccharomyces cerevisiae ALD5 NP_010996.2 330443526 Saccharomycescerevisiae ALD6 ABX39192.1 160415767 Saccharomyces cerevisiae HFD1Q04458.1 2494079 Saccharomyces cerevisiae GOR1 NP_014125.1 6324055Saccharomyces cerevisiae YPL113C AAB68248.1 1163100 Saccharomycescerevisiae GCY1 CAA99318.1 1420317 Saccharomyces cerevisiae

Enzymes exhibiting 4-hydroxybutyrate dehydrogenase activity (EC1.1.1.61) also fall into this category. Such enzymes have beencharacterized in Ralstonia eutrophy (Bravo et al., J Forens Sci,49:379-387 (2004)), Clostridium kluyveri (Wolff et al., Protein Expr.Purif. 6:206-212 (1995)) and Arabidopsis thaliana (Breitkreuz et al., JBiol Chem, 278:41552-41556 (2003)). The A. thaliana enzyme was clonedand characterized in yeast (Breitkreuz et al., J. Biol. Chem.278:41552-41556 (2003)). Yet another gene is the alcohol dehydrogenaseadhI from Geobacillus thermoglucosidasius Peon et al., J Biotechnol135:127-133 (2008)).

Protein GenBank ID GI Number Organism 4hbd YP_726053.1 113867564Ralstonia eutropha H16 4hbd L21902.1 146348486 Clostridium kluyveri DSM555 4hbd Q94B07 75249805 Arabidopsis thaliana adhI AAR91477.1 40795502Geobacillus thermoglucosidasius

Another exemplary enzyme is methylmalonate semialdehyde reductase, alsoknown as 3-hydroxyisobutyrate dehydrogenase (EC 1.1.1.31). This enzymeparticipates in valine, leucine and isoleucine degradation and has beenidentified in bacteria, eukaryotes, and mammals. The enzyme encoded byP84067 from Thermus thermophilus HB8 has been structurally characterized(Lokanath et al., J Mol Biol, 352:905-17 (2005)). The reversibility ofthe human 3-hydroxyisobutyrate dehydrogenase was demonstrated usingisotopically-labeled substrate (Manning et al., Biochem J, 231:481-4(1985)). Additional genes encoding this enzyme include 3hidh in Homosapiens (Hawes et al., Methods Enzymol, 324:218-228 (2000)) andOryctolagus cuniculus (Hawes et al., supra; Chowdhury et al., Biosci.Biotechnol Biochem. 60:2043-2047 (1996)), mmsB in Pseudomonas aeruginosaand Pseudomonas putida, and dhat in Pseudomonas putida (Aberhart et al.,J Chem. Soc. [Perkin 1] 6:1404-1406 (1979); Chowdhury et al., Biosci.Biotechnol Biochem. 60:2043-2047 (1996); Chowdhury et al., Biosci.Biotechnol Biochem. 67:438-441 (2003)). Several 3-hydroxyisobutyratedehydrogenase enzymes have been characterized in the reductivedirection, including mmsB from Pseudomonas aeruginosa (Gokarn et al.,U.S. Pat. No. 739,676, (2008)) and mmsB from Pseudomonas putida.

Protein GenBank ID GI Number Organism P84067 P84067 75345323 Thermusthermophilus 3hidh P31937.2 12643395 Homo sapiens 3hidh P32185.1 416872Oryctolagus cuniculus mmsB NP_746775.1 26991350 Pseudomonas putida mmsBP28811.1 127211 Pseudomonas aeruginosa dhat Q59477.1 2842618 PseudomonasputidaFIG. 3, Step E—Dihydroxyacetone Phosphate Phosphatase orDihydroxyacetone Kinase

Kinase or phosphotransferase enzymes in the EC class 2.7.2 transformcarboxylic acids to phosphonic acids with concurrent hydrolysis of oneATP. Such an enzyme is required to form dihydroxyacetone fromdihydroxyacetone phosphate and glycerol from glycerol-3-phosphate.Exemplary dihydroxyacetone kinases enzymes (EC 2.7.2.29) include DAKfrom Pichia angusta and dhaK from Citrobacter freundii (van der Klei etal, Curr Genet, 34:1-11 (1998); Daniel et al., J Bacteriol, 177:4392-401(1995)). The E. coli enzyme is encoded by dhaKLM (Bachler et al, EMBO J,24: 283-93 (2005)).

Protein GenBank ID GI Number Organism dhaK AAC74284.2 87081857Escherichia coli dhaL AAC74283.1 1787449 Escherichia coli dhaMNP_415717.1 16129162 Escherichia coli DAK AAC27705.1 3171001 Pichiaangusta dhaK AAB48843.1 493083 Citrobacter freundii

The conversion of dihydroxyacetone phosphate to dihydroxyacetone iscatalyzed by dihydroxyacetone phosphate phosphatase. The acidphosphatase of Xenopus laevis was shown to catalyze the hydrolysis of awide range of acid phosphatases including dihydroxyacetone phosphate(Filburn, Arch Biochem Biophys 159:683-93 (1973)). The gene associatedwith this enzyme is not known.

Other suitable enzyme candidates for hydrolyzingglyceraldehydes-3-phosphate or dihydroxyacetone phosphate is3-phosphoglycerate phosphatase (EC 3.1.3.38), catalyzing the hydrolysisof 3PG to glycerate. The enzyme is found in plants and has a broadsubstrate range that includes phosphoenolpyruvate,ribulose-1,5-bisphosphate, dihydroxyacetone phosphate andglucose-6-phosphate (Randall et al., Plant Physiol 48:488-492 (1971);Randall et al., J Biol. Chem. 246:5510-5517 (1971)). Purified enzymefrom various plant sources has been characterized but a gene has notbeen associated with this enzyme to date. Another enzyme with3-phosphoglycerate phosphatase activity is the phosphoglyceratephosphatase (EC 3.1.3.20) from pig liver (Fallon et al., Biochim.Biophys. Acta 105:43-53 (1965)). The gene associated with this enzyme isnot available.

The enzyme alkaline phosphatase (EC 3.1.3.1) hydrolyses a broad range ofphosphorylated substrates to their corresponding alcohols. These enzymesare typically secreted into the periplasm in bacteria, where they play arole in phosphate transport and metabolism. The E. coli phoA geneencodes a periplasmic zinc-dependent alkaline phosphatase active underconditions of phosphate starvation (Coleman Annu. Rev. Biophys. Biomol.Struct. 21:441-83 (1992)). Similar enzymes have been characterized inCampylobacter jejuni (van Mourik et al., Microbiol. 154:584-92 (2008)),Saccharomyces cerevisiae (Oshima et al., Gene 179:171-7 (1996)) andStaphylococcus aureus (Shah and Blobel, J. Bacteriol. 94:780-1 (1967)).Enzyme engineering and/or removal of targeting sequences may be requiredfor alkaline phosphatase enzymes to function in the cytoplasm.

Protein GenBank ID GI Number Organism phoA NP_414917.2 49176017Escherichia coli phoX ZP_01072054.1 86153851 Campylobacter jejuni PHO8AAA34871.1 172164 Saccharomyces cerevisiae SaurJH1_2706 YP_001317815.1150395140 Staphylococcus aureusFIG. 3, Step F—Dihydroxyacetone Reductase

Dihydroxyacetone reductase (EC 1.1.1.6 and 1.1.1.156) or glyceroldehydrogenase enzymes have been characterized in numerous organisms. Anexemplary NADPH-dependent dihydroxyacetone reductase is encoded by gld2of Hypocrea jecorina (Liepins et al, FEBS J 273:4229-4235 (2006)).NADH-dependent enzymes include gldA of E. coli and dhaD of Klebsiellapneumoniae (Altaras and Cameron., Appl Env Microbiol., 65:1180-1185(1999)). The glycerol dehydrogenase GCY1 of Saccharomyces cerevisiae isalso suitable here (WO 2011/022651A1).

Protein GenBank ID GI Number Organism Gld2 Q0GYU4 121924008 Hypocreajecorina GCY1 CAA99318.1 1420317 Saccharomyces cerevisiae gldAAAC76927.2 87082352 Escherichia coli dhaD ABO15720.1 126513217Klebsiella pneumoniaeFIG. 3, Step G—Dihydroxyacetone Phosphate Reductase

The reduction of dihydroxyacetone phosphate to glycerol-3-phosphate iscatalyzed by numerous enzymes including glycerol-3-phosphatedehydrogenase, glycerol dehydrogenase, galactitol 2-dehydrogenaseD-xylulose reductase and glycerol-1-phosphate dehydrogenase. Exemplaryglycerol-3-phosphate dehydrogenase enzymes with demonstrateddihydroxyacetone phosphate reductase activity include GPD1 ofSaccharomyces cerevisiae (Carnbon et al, Appl Environ Microbiol72:4688-94 (2006)) and GPDH of Osmerus mordax (Liebscher et al, PhysiolBiochem Zool 79:411-23 (2006)). The galactitol dehydrogenase ofRhodobacter sphaeroides also has this activity (Carius et al, J BiolChem, 25:20006-14 (2010)). The glycerol dehydrogenase enzyme ofEmericella nidulans also catalyzes this reaction (Schuurink et al, J GenMicrobiol 136:1043-50 (1990)).

Protein GenBank ID GI Number Organism GPD1 CAA98582.1 1430995Saccharomyces cerevisiae GatDH ACM89305.1 223413895 Rhodobactersphaeroides gldB Q7Z8L1 74619179 Emericella nidulans

4.4 Example IV—Methods of Using Formaldehyde Produced from the Oxidationof Methanol in the Formation of Intermediates of Central MetabolicPathways for the Formation of Biomass

Provided herein are exemplary pathways, which utilize formaldehydeproduced from the oxidation of methanol (see, e.g., FIG. 1, step J) inthe formation of intermediates of certain central metabolic pathwaysthat can be used for the formation of biomass. Exemplary methanolmetabolic pathways for enhancing the availability of reducingequivalents, as well as the producing formaldehyde from methanol (stepJ), are provided in FIG. 1.

One exemplary pathway that can utilize formaldehyde produced from theoxidation of methanol (e.g., as provided in FIG. 1) is shown in FIG. 4,which involves condensation of formaldehyde and D-ribulose-5-phosphateto form hexylose-6-phosphate (h6p) by hexylose-6-phosphate synthase(FIG. 4, step A). The enzyme can use Mg²⁺ or Mn²⁺ for maximal activity,although other metal ions are useful, and even non-metal-ion-dependentmechanisms are contemplated. H6p is converted into fructose-6-phosphateby 6-phospho-3-hexyloisomerase (FIG. 4, step B).

Another exemplary pathway that involves the detoxification andassimilation of formaldehyde produced from the oxidation of methanol(e.g., as provided in FIG. 1) is shown in FIG. 5 and proceeds throughdihydroxyacetone. Dihydroxyacetone synthase is a special transketolasethat first transfers a glycoaldehyde group from xylulose-5-phosphate toformaldehyde, resulting in the formation of dihydroxyacetone (DHA) andglyceraldehyde-3-phosphate (G3P), which is an intermediate in glycolysis(FIG. 5, step A). The DHA obtained from DHA synthase is then furtherphosphorylated to form DHA phosphate by a DHA kinase (FIG. 5, step B).DHAP can be assimilated into glycolysis and several other pathways.

FIG. 4, Steps A and B—Hexylose-6-phosphate synthase (Step A) and6-phospho-3-hexyloisomerase (Step B)

Both of the hexylose-6-phosphate synthase and6-phospho-3-hexyloisomerase enzymes are found in several organisms,including methanotrops and methylotrophs where they have been purified(Kato et al., 2006, BioSci Biotechnol Biochem. 70(1):10-21. In addition,these enzymes have been reported in heterotrophs such as Bacillussubtilis also where they are reported to be involved in formaldehydedetoxification (Mitsui et al., 2003, AEM 69(10):6128-32, Yasueda et al.,1999. J Bac 181(23):7154-60. Genes for these two enzymes from themethylotrophic bacterium Mycobacterium gastri MB19 have been fused andE. coli strains harboring the hps-phi construct showed more efficientutilization of formaldehyde (Orita et al. 2007, Appl MicrobiolBiotechnol. 76:439-445). In some organisms, these two enzymes naturallyexist as a fused version that is bifunctional.

Exemplary candidate genes for hexylose-6-phopshate synthase are:

Protein GenBank ID GI number Organism Hps AAR39392.1 40074227 Bacillusmethanolicus MGA3 Hps EIJ81375.1 387589055 Bacillus methanolicus PB1RmpA BAA83096.1 5706381 Methylomonas aminofaciens RmpA BAA90546.16899861 Mycobacterium gastri YckG BAA08980.1 1805418 Bacillus subtilis

Exemplary gene candidates for 6-phospho-3-hexyloisomerase are:

Protein GenBank ID GI number Organism Phi AAR39393.1 40074228 Bacillusmethanolicus MGA3 Phi EIJ81376.1 387589056 Bacillus methanolicus PB1 PhiBAA83098.1 5706383 Methylomonas aminofaciens RmpB BAA90545.1 6899860Mycobacterium gastri

Candidates for enzymes where both of these functions have been fusedinto a single open reading frame include the following.

Protein GenBank ID GI number Organism PH1938 NP_143767.1 14591680Pyrococcus horikoshii OT3 PF0220 NP_577949.1 18976592 Pyrococcusfuriosus TK0475 YP_182888.1 57640410 Thermococcus kodakaraensisNP_127388.1 14521911 Pyrococcus abyssi MCA2738 YP_115138.1 53803128Methylococcus capsulatasFIG. 5, Step A—Dihydroxyacetone Synthase

Another exemplary pathway that involves the detoxification andassimilation of formaldehyde produced from the oxidation of methanol(e.g., as provided in FIG. 1) is shown in FIG. 5 and proceeds throughdihydroxyacetone. Dihydroxyacetone synthase is a special transketolasethat first transfers a glycoaldehyde group from xylulose-5-phosphate toformaldehyde, resulting in the formation of dihydroxyacetone (DHA) andglyceraldehyde-3-phosphate (G3P), which is an intermediate in glycolysis(FIG. 5, step A). The DHA obtained from DHA synthase is then furtherphosphorylated to form DHA phosphate by a DHA kinase (FIG. 5, step B).DHAP can be assimilated into glycolysis and several other pathways.

The dihydroxyacetone synthase enzyme in candida boidinii uses thiaminepyrophosphate and Mg²⁺ as cofactors and is localized in the peroxisome.The enzyme from the methanol-growing carboxydobacterium, Mycobacter sp.strain JC1 DSM 3803, was also found to have DHA synthase and kinaseactivities (Ro et al., 1997, JBac 179(19):6041-7). DHA synthase fromthis organism also has similar cofactor requirements as the enzyme fromC. boidinii. The K_(m)s for formaldehyde and xylulose 5-phosphate werereported to be 1.86 mM and 33.3 microM, respectively. Several othermycobacteria, excluding only Mycobacterium tuberculosis, can usemethanol as the sole source of carbon and energy and are reported to usedihydroxyacetone synthase (Part et al., 2003, JBac 185(1):142-7.

Protein GenBank ID GI number Organism DAS1 AAC83349.1 3978466 Candidaboidinii HPODL_2613 EFW95760.1 320581540 Ogataea parapolymorpha DL-1(Hansenula polymorpha DL-1) AAG12171.2 18497328 Mycobacter sp. strainJC1 DSM 3803FIG. 5, Step B—Dihydroxyacetone (DHA) Kinase

DHA obtained from DHA synthase is further phosphorylated to form DHAphosphate by a DHA kinase. DHAP can be assimilated into glycolysis andseveral other pathways. Dihydroxyacetone kinase has been purified fromOgataea angusta to homogeneity (Bystrkh, 1983, Biokhimiia,48(10):1611-6). The enzyme, which phosphorylates dihydroxyacetone and,to a lesser degree, glyceraldehyde, is a homodimeric protein of 139 kDa.ATP is the preferred phosphate group donor for the enzyme. When ITP,GTP, CTP and UTP are used, the activity drops to about 30%. In severalorganisms such as Klebsiella pneumoniae and Citrobacter fruendii (Danielet al., 1995, JBac 177(15):4392-40), DHA is formed as a result ofoxidation of glycerol and is converted into DHAP by the kinase DHAkinase of K. pneumoniae has been characterized (Jonathan et al, 1984,JBac 160(1):55-60). It is very specific for DHA, with a K_(m) of 4 μM,and has two apparent K_(m) values for ATP, one at 25 to 35 μM, and theother at 200 to 300 μM. DHA can also be phosphorylated by glycerolkinases but the DHA kinase from K. puemoniae is different from glycerolkinase in several respects. While both enzymes can phosphorylatedihydroxyacetone, DHA kinase does not phosphorylate glycerol, neither isit inhibited by fructose-1,6-diphosphate. in Saccharomyces cerevisiae,DHA kinases (I and II) are involved in rescuing the cells from toxiceffects of dihydroxyacetone (Molin et al., 2003, J Biol Chem. 17;278(3):1415-23).

In Escherichia coli, DHA kinase is composed of the three subunits DhaK,DhaL, and DhaM and it functions similarly to a phosphotransferase system(PTS) in that it utilizes phosphoenolpyruvate as a phosphoryl donor(Gutknecht et al., 2001, EMBO J. 20(10):2480-6). It differs in not beinginvolved in transport. The phosphorylation reaction requires thepresence of the EI and HPr proteins of the PTS system. The DhaM subunitis phosphorylated at multiple sites. DhaK contains the substrate bindingsite (Garcia-Alles et al., 2004, 43(41):13037-45; Siebold et al., 2003,PNAS. 100(14):8188-92). The K_(M) for dihydroxyacetone for the E. colienzyme has been reported to be 6 μM. The K subunit is similar to theN-terminal half of ATP-dependent dihydroxyacetone kinase of Citrobacterfreundii and eukaryotes.

Exemplary DHA kinase gene candidates for this step are:

Protein GenBank ID GI number Organism DAK1 P54838.1 1706391Saccharomyces cerevisiae S288c DAK2 P43550.1 1169289 Saccharomycescerevisiae S288c D186_20916 ZP_16280678.1 421847542 Citrobacter freundiiDAK2 ZP_18488498.1 425085405 Klebsiella pneumoniae DAK AAC27705.13171001 Ogataea angusta DhaK NP_415718.6 162135900 Escherichia coli DhaLNP_415717.1 16129162 Escherichia coli DhaM NP_415716.4 226524708Escherichia coli

4.5 Example V—Methods for Handling Anaerobic Cultures

This example describes methods used in handling anaerobic cultures.

A. Anaerobic Chamber and Conditions.

Exemplary anaerobic chambers are available commercially (see, forexample, Vacuum Atmospheres Company, Hawthorne Calif.; MBraun,Newburyport Mass.). Conditions included an O₂ concentration of 1 ppm orless and 1 atm pure N₂. In one example, 3 oxygen scrubbers/catalystregenerators were used, and the chamber included an O₂ electrode (suchas Teledyne; City of Industry Calif.). Nearly all items and reagentswere cycled four times in the airlock of the chamber prior to openingthe inner chamber door. Reagents with a volume>5 mL were sparged withpure N₂ prior to introduction into the chamber. Gloves are changedtwice/yr and the catalyst containers were regenerated periodically whenthe chamber displays increasingly sluggish response to changes in oxygenlevels. The chamber's pressure was controlled through one-way valvesactivated by solenoids. This feature allowed setting the chamberpressure at a level higher than the surroundings to allow transfer ofvery small tubes through the purge valve.

The anaerobic chambers achieved levels of O₂ that were consistently verylow and were needed for highly oxygen sensitive anaerobic conditions.However, growth and handling of cells does not usually require suchprecautions. In an alternative anaerobic chamber configuration, platinumor palladium can be used as a catalyst that requires some hydrogen gasin the mix. Instead of using solenoid valves, pressure release can becontrolled by a bubbler. Instead of using instrument-based O₂monitoring, test strips can be used instead.

B. Anaerobic Microbiology.

Serum or media bottles are fitted with thick rubber stoppers andaluminum crimps are employed to seal the bottle. Medium, such asTerrific Broth, is made in a conventional manner and dispensed to anappropriately sized serum bottle. The bottles are sparged with nitrogenfor ˜30 min of moderate bubbling. This removes most of the oxygen fromthe medium and, after this step, each bottle is capped with a rubberstopper (such as Bellco 20 mm septum stoppers; Bellco, Vineland, N.J.)and crimp-sealed (Bellco 20 mm). Then the bottles of medium areautoclaved using a slow (liquid) exhaust cycle. At least sometimes aneedle can be poked through the stopper to provide exhaust duringautoclaving; the needle needs to be removed immediately upon removalfrom the autoclave. The sterile medium has the remaining mediumcomponents, for example buffer or antibiotics, added via syringe andneedle. Prior to addition of reducing agents, the bottles areequilibrated for 30-60 minutes with nitrogen (or CO depending upon use).A reducing agent such as a 100×150 mM sodium sulfide, 200 mMcysteine-HCl is added. This is made by weighing the sodium sulfide intoa dry beaker and the cysteine into a serum bottle, bringing both intothe anaerobic chamber, dissolving the sodium sulfide into anaerobicwater, then adding this to the cysteine in the serum bottle. The bottleis stoppered immediately as the sodium sulfide solution generateshydrogen sulfide gas upon contact with the cysteine. When injecting intothe culture, a syringe filter is used to sterilize the solution. Othercomponents are added through syringe needles, such as B12 (10 μMcyanocobalamin), nickel chloride (NiCl₂, 20 microM final concentrationfrom a 40 mM stock made in anaerobic water in the chamber and sterilizedby autoclaving or by using a syringe filter upon injection into theculture), and ferrous ammonium sulfate (final concentration needed is100 μM—made as 100-1000× stock solution in anaerobic water in thechamber and sterilized by autoclaving or by using a syringe filter uponinjection into the culture). To facilitate faster growth under anaerobicconditions, the 1 liter bottles were inoculated with 50 mL of apreculture grown anaerobically. Induction of the pA1-lacO1 promoter inthe vectors was performed by addition of isopropylβ-D-1-thiogalactopyranoside (IPTG) to a final concentration of 0.2 mMand was carried out for about 3 hrs.

Large cultures can be grown in larger bottles using continuous gasaddition while bubbling. A rubber stopper with a metal bubbler is placedin the bottle after medium addition and sparged with nitrogen for 30minutes or more prior to setting up the rest of the bottle. Each bottleis put together such that a sterile filter will sterilize the gasbubbled in and the hoses on the bottles are compressible with small Cclamps. Medium and cells are stirred with magnetic stir bars. Once allmedium components and cells are added, the bottles are incubated in anincubator in room air but with continuous nitrogen sparging into thebottles.

Throughout this application various publications have been referenced.The disclosures of these publications in their entireties, includingGenBank and GI number publications, are hereby incorporated by referencein this application in order to more fully describe the state of the artto which this invention pertains. Although the invention has beendescribed with reference to the examples and embodiments provided above,it should be understood that various modifications can be made withoutdeparting from the spirit of the invention.

What is claimed is:
 1. A non-naturally occurring microbial organismcomprising: (A) a methanol metabolic pathway, wherein said non-naturallyoccurring microbial organism comprises at least one exogenous nucleicacid encoding a methanol metabolic pathway enzyme expressed in asufficient amount to enhance the availability of reducing equivalents inthe presence of methanol or metabolize methanol as a carbon source forbiosynthesis of 1,2-propanediol or n-propanol, wherein said methanolmetabolic pathway comprises: (i) a methanol methyltransferase and amethylenetetrahydrofolate reductase; (ii) a methanol dehydrogenase; or(iii) a methanol dehydrogenase and a formaldehyde activating enzyme; andone of: (B) a 1,2-propanediol pathway wherein the non-naturallyoccurring microbial organism comprises at least one exogenous nucleicacid encoding a 1,2-propanediol pathway enzyme expressed in a sufficientamount to produce 1,2-propanediol, wherein said 1,2-propanediol pathwaycomprises the 1,2 propanediol pathway enzymes: (i) 2A, 2B, and 2C; or(ii) 2A, 2D, and 2E; or (C) a n-propanol pathway wherein thenon-naturally occurring microbial organism comprises at least oneexogenous nucleic acid encoding a n-propanol pathway enzyme expressed ina sufficient amount to produce n-propanol, wherein said n-propanolpathway comprises n-propanol pathway enzymes: (i) 2A, 2B, 2C, 2F, and2G; or (ii) 2A, 2D, 2E, 2F, and 2G; wherein 2A is a methylglyoxalsynthase that enzymatically converts dihydroxyacetone phosphate tomethylglyoxal, 2B is a methylglyoxal reductase (acetol-forming) thatenzymatically converts methylglyoxal to acetol, 2C is an acetolreductase that enzymatically converts acetol to 1,2-propanediol, 2D is amethylglyoxal reductase (lactaldehyde-forming) that enzymaticallyconverts methylglyoxal to lactaldehyde, 2E is a lactaldehyde reductasethat converts lactaldehyde to 1,2-propanediol, 2F is a 1,2-propanedioldehydratase that converts 1,2-propanediol to propanal, and 2G is apropanal reductase that converts propanal to propanol.
 2. Thenon-naturally occurring microbial organism of claim 1, wherein thenon-naturally occurring microbial organism comprises two or threeexogenous nucleic acids, each encoding a 1,2-propanediol pathway enzyme.3. The organism of claim 1, wherein the methanol metabolic pathwaycomprises: (i) a methanol methyltransferase, a methylenetetrahydrofolatereductase, a methylenetetrahydrofolate dehydrogenase, amethenyltetrahydrofolate cyclohydrolase, and a formyltetrahydrofolatedeformylase; (ii) a methanol methyltransferase, amethylenetetrahydrofolate reductase, a methylenetetrahydrofolatedehydrogenase, a methenyltetrahydrofolate cyclohydrolase and aformyltetrahydrofolate synthetase; (iii) a methanol dehydrogenase, amethylenetetrahydrofolate dehydrogenase, a methenyltetrahydrofolatecyclohydrolase and a formyltetrahydrofolate deformylase; (iv) a methanoldehydrogenase, a methylenetetrahydrofolate dehydrogenase, amethenyltetrahydrofolate cyclohydrolase and a formyltetrahydrofolatesynthetase; (v) a methanol dehydrogenase and a formaldehydedehydrogenase; (vi) a methanol dehydrogenase, aS-(hydroxymethyl)glutathione synthase, a glutathione-dependentformaldehyde dehydrogenase and a S-formylglutathione hydrolase; (vii) amethanol dehydrogenase, a glutathione-dependent formaldehydedehydrogenase and a S-formylglutathione hydrolase; (viii) a methanoldehydrogenase, a formaldehyde activating enzyme, amethylenetetrahydrofolate dehydrogenase, a methenyltetrahydrofolatecyclohydrolase and a formyltetrahydrofolate deformylase; or (ix) amethanol dehydrogenase, a formaldehyde activating enzyme, amethylenetetrahydrofolate dehydrogenase, a methenyltetrahydrofolatecyclohydrolase and a formyltetrahydrofolate synthetase.
 4. Thenon-naturally occurring microbial organism of claim 1, wherein: saidnon-naturally occurring microbial organism comprises two, three, four,five, six or seven exogenous nucleic acids, each encoding a methanolmetabolic pathway enzyme.
 5. The non-naturally occurring microbialorganism of claim 1, further comprising a formaldehyde assimilationpathway, wherein said non-naturally occurring microbial organismcomprises at least one exogenous nucleic acid encoding a formaldehydeassimilation pathway enzyme expressed in a sufficient amount to producean intermediate of glycolysis and/or a metabolic pathway that can beused in the formation of biomass, and wherein said formaldehydeassimilation pathway comprises: (A) a hexulose-6-phosphate synthase anda 6-phospho-3-hexuloisomerase; or (B) a dihydroxyacetone synthase and adihydroxyacetone kinase.
 6. The non-naturally occurring microbialorganism of claim 1, wherein-said organism is a species of bacteria,yeast, or fungus.
 7. A method for producing 1,2-propanediol orn-propanol; the method comprising culturing the respective non-naturallyoccurring microbial organism of claim 1 under conditions and for asufficient period of time to produce 1,2-propanediol or n-propanol. 8.The non-naturally occurring microbial organism of claim 1, wherein theat least one exogenous nucleic acid encoding a methanol metabolicpathway enzyme is a heterologous nucleic acid.
 9. The non-naturallyoccurring microbial organism of claim 1, wherein the at least oneexogenous nucleic acid encoding a 1,2-propanediol pathway enzyme is aheterologous nucleic acid.
 10. The non-naturally occurring microbialorganism of claim 1, wherein the non-naturally occurring microbialorganism comprises two, three, four or five exogenous nucleic acids,each encoding a n-propanol pathway enzyme.
 11. The non-naturallyoccurring microbial organism of claim 1, wherein the at least oneexogenous nucleic acid encoding a n-propanol pathway enzyme is aheterologous nucleic acid.
 12. The non-naturally occurring microbialorganism of claim 1, wherein the non-naturally occurring microbialorganism comprises one or more gene disruptions, the one or more genedisruptions occurring in one or more endogenous genes encodingprotein(s) or enzyme(s) involved in native production of ethanol,glycerol, acetate, lactate, formate, CO₂, and/or amino acids, by thenon-naturally occurring microbial organism, and wherein the one or moregene disruptions confer(s) increased production of 1,2-propanediol orn-propanol in the non-naturally occurring microbial organism.
 13. Thenon-naturally occurring microbial organism of claim 12, wherein the oneor more endogenous enzymes involved in native production of ethanol,glycerol, acetate, lactate, formate, CO₂ and/or amino acids by saidmicrobial organism have attenuated enzyme activity or expression levels.14. The non-naturally occurring microbial organism of claim 3, whereinthe methanol metabolic pathway further comprises: (A) a formatedehydrogenase; (B) a formate hydrogen lyase; or (C) a formate hydrogenlyase and a hydrogenase.
 15. The non-naturally occurring microbialorganism of claim 5, wherein the intermediate of glycolysis and/or ametabolic pathway is: (A) a hexulose-6-phosphate, afructose-6-phosphate, or a combination thereof; or (B) adihydroxyacetone, a dihydroxyacetone phosphate, or a combinationthereof.
 16. The non-naturally occurring microbial organism of claim 5,wherein the non-naturally occurring microbial organism comprises twoexogenous nucleic acids, each encoding a formaldehyde assimilationpathway enzyme.
 17. The non-naturally occurring microorganism of claim6, wherein the non-naturally occurring microbial organism is selectedfrom the group consisting of: (A) Escherichia coli, Klebsiella oxytoca,Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes,Mannheimia succiniciproducens, Rhizobium etli, Bacillus subtilis,Corynebacterium glutamicum, Gluconobacter oxydans, Zymomonas mobilis,Lactococcus lactis, Lactobacillus plantarum, Streptomyces coelicolor,Clostridium acetobutylicum, Pseudomonas fluorescens, and Pseudomonasputida; or (B) Saccharomyces cerevisiae, Schizosaccharomyces pombe,Kluyveromyces lactis, Kluyveromyces marxianus, Aspergillus terreus,Aspergillus niger, Pichia pastoris, Rhizopus arrhizus, and Rhizopusoryzae.
 18. The method of claim 7, wherein the method further comprisesseparating the 1,2-propanediol or n-propanol from other components inthe culture.
 19. The method of claim 18, wherein the separationcomprises extraction, continuous liquid-liquid extraction,pervaporation, membrane filtration, membrane separation, reverseosmosis, electrodialysis, distillation, crystallization, centrifugation,extractive filtration, ion exchange chromatography, size exclusionchromatography, adsorption chromatography, or ultrafiltration.
 20. Themethod of claim 7, wherein the non-naturally occurring microbialorganism is cultured in a medium comprising biomass, glucose, xylose,arabinose, galactose, mannose, fructose, sucrose, starch, glycerol,methanol, carbon dioxide, formate, methane, or any combination thereofas a carbon source.
 21. The method of claim 7, wherein the non-naturallyoccurring microbial organism is cultured in a medium comprising methanolas a carbon source.
 22. The method of claim 7, wherein the non-naturallyoccurring microbial organism is cultured in a medium consistingessentially of methanol as a carbon source.
 23. The method of claim 7,wherein the non-naturally occurring microbial organism is cultured in asubstantially anaerobic culture medium.